A novel design of gene therapy carriers - pH sensitive cationic nanoparticles with encapsulated iron oxide particles

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UPTEC X 010 27

Examensarbete 20 p November 2010

A novel design of gene therapy carriers - pH sensitive cationic nanoparticles with encapsulated iron oxide particles

Jens Roat Kultima

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Bioinformatics Engineering Program

Uppsala University School of Engineering UPTEC X 10 27 Date of issue 2010-11

Author

Jens Roat Kultima

Title (English)

A novel design of gene therapy carriers - pH sensitive cationic nanoparticles with encapsulated iron oxide particles

Title (Swedish)

Abstract

Here we present a gene delivery carrier, consisting of cationic pH sensitive nanoparticles with encapsulated iron oxide particles. We have been able to control the size and magnetic loading of these nanoparticles. The dissolution profiles at different pH’s has been established and it has been shown that these particles have potential as gene delivery carriers.

Keywords

Eudragit, E PO, L 100, L100-55, S 100, PLGA, nanoparticles, iron oxide, gene therapy, pH sensitive particles

Supervisors

Jeffrey M. Karp, Ph.D.

Chenjie Xu, Ph.D.

Center for Regenerative Therapeutics & Deptartment of Medicine, BWH Harvard Medical School, Harvard Stem Cell Institute,

Harvard-MIT Division of Health Science and Technology

Scientific reviewer

Jöns Hilborn, Ph.D.

Uppsala universitet

Project name Sponsors

Language

English

Security

2014-11

ISSN 1401-2138 Classification

Supplementary bibliographical information Pages

40

Biology Education Centre Biomedical Center Husargatan 3 Uppsala Box 592 S-75124 Uppsala Tel +46 (0)18 4710000 Fax +46 (0)18 471 4687

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M.SC. THESIS

A novel design of gene therapy carriers - pH sensitive cationic nanoparticles with encapsulated iron oxide particles

JENS ROATKULTIMA¹⁻⁵, B.Sc.

Supervisors

JEFFREY M. KARP²⁻⁵, Ph.D.

CHENJIEXU²⁻⁵, Ph.D.

Scientific Reviewer JÖNSHILBORN¹, Ph.D.

Examinator

MARGARETAKRABBE¹, Ph.D.

1Uppsala University

2Center for Regenerative Therapeutics &

Department of Medicine, Brigham & Women’s Hospital

3Harvard Medical School

4Harvard Stem Cell Institute

5Harvard-MIT Division of Health Science and Technology

November 29, 2010

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JENSROATKULTIMA, B.Sc.

Abstract

Gene therapy has been one of the hottest research fields in the last decade. Focus has been mainly on developing DNA plasmids to be delivered to cells, and methods of delivering these plasmids. Different types of vectors have been developed, with an initial focus on viral vectors. Due to cost, toxicity and adverse immune problems of these vessels, focus has shifted more towards nonviral delivery systems and specifically different types of nanoparticles with high biocompatibility and potential for large-scale production. There are several barriers a potential DNA carrier must overcome: avoid clearance by the reticuloendothelial system, vessel delivery and adhesion to the cell surface, entry into the cell, endosomal escape and finally nuclear translocation of conjugated DNA. Surface modification with hydrophilic polymers or plasma protein can prevent clearance form the reticuloendothelial system. Magnetism has been proposed to control particle localization to organs, tissues and cells. It has been shown that cellular uptake of DNA varies significantly based on carrier vector.

Nanoparticles have been modified by adding cationic macromolecules onto the surface of these particles to maximize the efficiency of gene transfection. Once the nanoparticles have reached the inside of the cell through endocytois, the endosomal wall has to be penetrated. This has been explored using several different methods;

such as using chloroquine, a well-known lysosomotropic agent or the incoorperation of membrane-destabilizing peptides. Here we present a homing gene delivery carrier, consisting of cationic pH sensitive nanoparticles with encapsulated iron oxide particles. The iron oxide particles enable the nanoparticles to be translocated to the cell surfaces through the use of an magnetic field. The cationic properties of the nanoparticle will enable cell surface adhesion. The pH sensitive nanoparticles will degrade once inside the cellular endosomes and quickly disrupt the endosomal walls, allowing conjugated DNA to be translocated into the cell nucleus. Our system will increase the uptake of particles, improve the translocation of gene vector from carrier particles into cytoplasm, and reduce the cost of cell transfection.

Keywords: Eudragit, E PO, L 100, L100-55, S 100, PLGA, nanoparticles, iron oxide particles, gene therapy, pH, pH sensitive particles

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JENSROATKULTIMA, B.Sc.

Populärvetenskaplig sammanfattning

I slutet av åttio- och början av nittiotalet kom de första vetenskapliga rapporterna som visar att det går att implantera funktionella varianter av defekta gener i celler och få dessa celler att uttrycka det protein som kodas av den funktionella genen. Detta var inledningen till begreppet genterapi, som i korthet går ut på att transfektera nytt funktionellt genetisk material till cellen och därmed förändra cellens uttryck av protiner. För att kunna transfektera celler effektivt måste dessa gener, eller DNA segment, effektivt föras in i målcellen. För att lösa det här problemet har forskare utvecklat olika metoder att inkapsla DNA i olika typer av bärare. En sådan typ av bärare kan vara nanopartiklar. Dessa nanopartiklar har tidigare bestått av t.ex.

guld eller silica. De första metoderna för att föra in partiklarna i målcellerna var att skjuta in dem. Senare metoder använder sig av cellens egna system för upptag, exempelvis genom endocytos. I det här arbetet presenterar vi en bärare som består av pH känsliga nanopartiklar uppbyggda av en pH känslig polymer med inkapslade järnoxidpartiklar, till vilka gener eller DNA fragment kan tillfogats genom adhesion. De syntetiserade nanopartiklarna upptas lätt av cellen och väl inne i cellerna bryts de ner snabbt. Sedan frigörs innehållet i partiklarna och detta kan sedan tas sig in i cellkärnan.

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To mom and dad for supporting me in everything I do.

To Elisabeth for always standing by my side.

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List of Tables

3.1 Synthesized Rhodamine 6G-loaded EPO particles centrifuged at 14,000 rpm for 5 min. . . . 24 3.2 Synthesized Rhodamine 6G-loaded EPO particles centrifuged

at 14,000 rpm for 20 min. . . . 24 3.3 Synthesized Rhodamine 6G-loaded S100, L100 and L100-55

particles centrifuged at 14,000 rpm for 5 min. . . . 25 3.4 Synthesized Rhodamine 6G-loaded PLGA particles. . . . 25 6.1 Synthesized Rhodamine 6G-loaded particles and their

respective size distributions, given by DLS and Coulter Counter (CC), if measured. . . . 36 7.1 Endosomal pH of non-activated and LPS activated MSCs and

RAW264.7 cells. . . . 41 8.1 Synthesized iron oxide- and iron oxide &DiO-loaded particles

and their respective size distributions, given by DLS. . . . 50

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List of Figures

1.1 Conceptual outline of the DNA delivery system. . . . 19 2.1 Structural formulae of the pH sensitive polymers. . . . 21 6.1 The effect of PVA concentration in the IAP, on average diam-

eter of Rhodamine 6G-loaded particles. . . . 35 6.2 Effect of IOP on average diameter of Rhodamine 6G-loaded

particles. . . . 35 6.3 Effect of centrifugation time on average diameter of

Rhodamine 6G-loaded particles. . . . 36 6.4 Typical size distribution of Rhodamine 6G-loaded EPO particles 37 6.5 Representative SEM images of Rhodamine 6G-loaded EPO,

S100 and PLGA particles . . . . 38 7.1 Dissolution profiles of Rhodamine 6G-loaded EPO particles

in different pH buffers. . . . 40 7.2 Dissolution profiles of Rhodamine 6G-loaded PLGA particles

in different pH buffers. . . . 41 7.3 Rhodamine 6G-loaded EPO particles, after 6 hours, incubated

in pH 4 and 7 buffer solutions. . . . 42 7.4 Calibration curves for dissolution profiles of Rhodamine 6G-

loaded EPO particles. . . . 43 7.5 Calibration curve for dissolution profiles of Rhodamine 6G-

loaded PLGA particles. . . . 43 7.6 The uptake efficiency of Rhodamine 6G-loaded EPO particles

for MSCs and RAW264.7 cells. . . . 44 7.7 Microsopic images of MSCs incubated with 0.5, 0.1 and 0.02

mg EPO particles / ml. . . . 45 7.8 Microsopic images of RAW264.7 cells incubated with 0.5, 0.1

and 0.02 mg EPO particles / ml. . . . 46 7.9 Digestion efficiency of Rhodamine 6G-loaded EPO and

PLGA particles for MSCs and RAW264.7 cells. . . . 47 7.10 Calibration curve for the dissolution profiles of Rhodamine

6G-loaded EPO and PLGA particles for the in vitro digestion experiment. . . . 47

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7.11 Calibration curves used to calculate pH values from measured fluorescence intensities. . . . 47 7.12 Microscopic images of RAW264.7 cells and MSCs incubated

with and without LPS. . . . 48 7.13 Microscopic images of MSCs incubated with different con-

centrations of Rhodamine 6G-loaded EPO particles at different time points. . . . 49 7.14 Microscopic images of MSCs incubated with different con-

centrations of Rhodamine 6G-loaded PLGA particles. . . . 49 8.1 TEM images of iron oxide- and iron oxide- & DiO-loaded

EPO particles. . . . 52 8.2 Zeta potential of iron oxide-loaded EPO and PLGA particles. . 53 8.3 FACS results of MSCs incubated without particles, with EPO

particles and also with EPO-PLL particles. . . . 53 8.4 Confocal microscopic images of MSCs and RAW264.7 cells

incubated with iron oxide- & DiO-loaded EPO particles. . . . . 54 8.5 Cell viability of MSCs and RAW264.7 cells, after incubating

cells with iron oxide- & DiO-loaded EPO particles for 3 hours. 55

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Nomenclature

D-E-I Dichloromethane-to-ethanol-to-isopropyl

DCM Dichloromethane

DCM-EtOH-ISP Dichloromethane-to-ethanol-to-isopropyl

DiD 1,1’-Dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine perchlorate

DiO C18 3,3’-dioctadecyloxacarbocyanine perchlorate

DLS Dynamic Light Scattering

DMEM Dulbeccos Modified Essential Medium

EAP external aqueous phase

EDTA ethylenediaminetetraacetic acid

EPO Eudragit E PO

EtOH Ethanol

FBS Fetal Bovine Serum

IAP internal aqueous phase

IOP internal organic phase

L-Glu L-Glutamine

L100 Eudragit L 100

L100-55 Eudragit L 100-55

LPS Lipopolysaccharide

MEM-alpha Minimum Essential Medium-Alpha

MeOH Methanol

mg milligram

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MSC Mesenchymal Stem Cells

MSC Multi-potent Stromal Cell

PEI Polyethylenimine

PLGA Poly(lactic-co-glycolic acid)

PLL Poly-L-Lysine

PVA Polyvinyl alcohol

RAW264.7 cells Mouse leukaemic monocyte macrophage cell line

rpm revolutions per minute

S100 Eudragit S 100

SDS Sodium Dodecyl Sulfate

siRNA small interfering RNA

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Index

Boric acid, 19 Citric acid, 19 Coulter Counter, 23 D-E-I, 21

Dichloromethane, 21 DiD, 29

DiO, 28 DMEM, 20

double-emulsion technique, 21 Dynamic Light Scattering, 23 EDTA, 20

Ethanol, 21 Eudragit E PO, 19 Eudragit L 100, 19 Eudragit L 100-55, 19 Eudragit S 100, 19

external aqueous phase, 21 Fe3O4, 19

Fetal Bovine Serum, 19 glycerol, 29

hexane, 19

Hydrochloric acid, 19 internal aqueous phase, 21 internal organic phase, 21 iron oxide particles, 19 L-Glutamine, 19 Lipopolysaccharide, 26 Lysotracker yellow/blue, 26 MEM-alpha, 19

Mesenchymal Stem Cells, 19

Methanol, 21

Multi-potent Stromal Cells, 19 Oleic acid, 19

pH sensitivity, 19 PLGA, 23

Poly-L-Lysine, 29 Polyethylenimine, 16 Polyvinyl alcohol, 21 RAW264.7 cells, 19 Rhodamine 6G, 21 SDS-HCl solution, 30 Simple PCI, 27 siRNA, 15

Sodium hydroxide, 19 Sodium phosphate, 19

sonication-emulsification method, 28 Tris buffer, 21

Trypsin, 20

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Part I:

Introduction

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1. Introduction

1.1 The birth of nanoparticle gene therapy

In the 80’s and 90’s, it was shown that diseases caused by a specific genetic defect could be cured by delivering a functioning copy of a defect gene (Fried- mann, 1996; Felgner et al., 1987; Hickman et al., 1994). These findings initi- ated the research field of gene therapy, which has been of increasing interested over the past decade (Gabhann et al., 2010; Duceppe and Tabrizian, 2010).

Research has mainly been focused on developing nucleic acids (DNA) to be delivered and methods of delivering the DNA (Patil et al., 2005).

Different types of vectors have been developed and employed for gene delivery; bacterial vectors (Darji et al., 1997; Loessner and Weiss, 2004), viral vectors (Felgner et al., 1987; Brunetti-Pierri and Ng, 2009; Blits et al., 2010) and other vectors (lipid-based, polymeric, dendrimer-based, polypep- tide and nanoparticles) (Mintzer and Simanek, 2009; Esposito et al., 1999;

Briones et al., 2001). Several specific disorders have been targets for gene therapy; severe combined immunodeficiency (Cavazzana-Calvo et al., 2000) and Parkinson⁰s disease (Kaplitt et al., 2007). Also tissue engineering, specifically using Multi-potent Stromal Cells (MSCs), has been a target for gene therapy (Goessler et al., 2006). The use of viral vectors in clinical applications, as gene delivery vehicles, has several limitations: toxicity, high cost, limited quality, cause of cell damage, and viral vectors can also induce adverse immune problems (Verma and Somia, 1997; Bergen et al., 2008; Mörner et al., 2009). Due to the limitations of viral vectors, research has focused more on other types of delivery vessels, such as polypeptides and nanoparticles. These compounds have high biocompatibility and potential for large-scale production (Behr, 1993). Nano- and microparticles can offer a number of advan- tages to other delivery systems: they are easily produced and stored, and also, they can be administered in different ways (oral, intramuscular, subcutaneous) (Yang et al., 2010; Esposito et al., 1999).

After administration, particles have to pass through endothelium or blood vessels to reach the target cells. Simultaneously, the particles have to pass through the reticuloendothelial system to avoid the clearance by B or T cells.

Once the particles reach the target cells, the most important steps are entry into the cell cytoplasm and release of DNA. Nanoparticles, with encapsulated DNA or DNA conjugated into the particle surface, can enter cells by endocytosis or phagocytosis. DNA can also be encapsulated inside particles and phys- ically shot into cells through electric charge. (Uchimura et al., 2007; Hauck 14

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et al., 2008; Mintzer and Simanek, 2009). Duceppe and Tabrizian (2010) states that "nanosized carriers are by far the most suitable delivery system for thera- peutic purposes".

1.2 In vitro barriers for cellular nanoparticle uptake

To achieve successful treatment of a disease using gene therapy, the therapeu- tic DNA must be delivered into the target cells. The DNA can be delivered with or without the use of a delivery vessel. Cellular uptake of free DNA via plasma membrane permeation is hindered by the size and negative charge of the DNA molecule and systemic circulation of DNA is hindered by nuclease degradation (Mintzer and Simanek, 2009). It has been shown that cellular uptake of DNA varies significantly based on carrier vector, cell type and surface glycosaminoglycans (GAGs) present on the cell and that the internalized complexes may be delivered into intracellular compartments that do not promote transcription (Ruponen et al., 2004). Nanoparticles have been modified by adding cationic macromolecules onto the nanoparticles to optimize delivery of genes (Andersen et al., 2010), by increasing the electrostatic differences between particle and cell surfaces. Ionic-ionic complexes formed by nega- tively charged DNA and positively charged nanoparticles have been used in both in vitro and in vivo studies to increase transfection of intact DNA (Zeng et al., 2010).

Cellular uptake of cationic complexes has been shown to proceed through different endocytic routes, such as clathrin-dependent endocytosis or by macropinocytosis, in different cell types (Kopatz et al., 2004; Gonçalves et al., 2004). The morphology of DNA complex formed with cationic polymers is independent of the polymer used, i.e. different cationic polymers interact similarly with the DNA to form complexes with similar surface properties and comparable uptake efficiencies. Though, the size of the formed nanoparticles effect the uptake efficiency (Mintzer and Simanek, 2009; Desai et al., 1997; Prabha et al., 2002). The optimal size of complexes for highest uptake is a mean diameter less than 200 nm. However, "enforced"

endocytosis may be promoted by changing the surface charge and thus allowing larger complexes with more surface area be internalized by the cell (Peer et al., 2007; Mintzer and Simanek, 2009; Aoyama et al., 2003). The theoretical optimal mean diameter of carriers for cellular uptake has been calculated to 54-60 nm (Gao et al., 2005).

Once the nanoparticles have reached the inside of the cell through endocytosis, they are located in lysosomes or endosomes. These compartments are more acidic (pH 5.0-6.4) than the cytosol or intracellular space (pH 7.4) (Ohkuma and Poole, 1978; Jiang et al., 1990). To transfer the DNA into the cell cytoplasm, the endosomal wall have to be penetrated. This has been explored using several different methods; such as using chloroquine, a well-

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known lysosomotropic agent that raises the pH of the endosome (de Duve et al., 1974), the incoorperation of membrane-destabilizing peptides (Wagner, 1999) or macromolecules with amine groups that exhibit "proton sponge" potential that buffer the endosomal vesicle leading to endosomal swelling and lysis. A rapid endosomal escape is desirable, and has been proven possible using modified poly(DL-lactide-co-glycolide) (PLGA) nanoparticles (Panyam et al., 2002). Once inside the cell, free DNA must enter the nucleus to initiate transcription and translation of the encoding genes.

Complexes less than 9-11 nm in diameter can passively enter the nucleus through pores in the nuclear membrane (Bonner, 1975), and larger structures enter the nucleus in an ATP-dependent process. This process is triggered by recognition of short peptide sequences and hindered by certain antinucleo- porin antibodies (Featherstone et al., 1988). Plasmid DNA can enter the nucleus more easily during cell division and positively charged vectors promote nuclear-localizing effect (Wilke et al., 1996; Pouton and Seymour, 2001).

Mintzer and Simanek (2009) concludes that "regardless of the exact method of nuclear entry, gene sequences complexed with cationic vector systems seem to have an advantage over free plasmid DNA for in vitro cell transfection".

1.3 Previous uses of nanoparticles as gene carriers

Early uses of nanoparticles to deliver DNA cells were based on shooting nanoparticles into cells, using acceleration devices. Tungsten nanoparticles were shoot inside cell using a gun powder explosion (Klein et al., 1992), and later gold nanoparticles were propelled into tissue by a helium gas shock wave (Williams et al., 1991). In both these system, the tissue was easily damaged by the external force applied. Gene transfer through this type of bombardment is applied to skin and used for genetic immunization applications (Larregina et al., 2001; Wang et al., 2004). More recent studies have focused on mod- ifying the surface of gold particles by adding cationic complexes, to allow for entry into the cell via endocytosis, rather than cell bombardment (Sandhu et al., 2002; Thomas and Klibanov, 2003; Noh et al., 2007).

Other particles used are surface-modified silica nanoparticles. These particles are inert, stable and relatively non-toxic (Kneuer et al., 2000; Sameti et al., 2003). Silica particles have been shown to enter the cells through the endocytic pathway, with an increased efficacy when using larger particles (Roy et al., 2005; Luo et al., 2004). A significant discovery in the field, was made by Bharali et al. (2005), when they, for the first time, showed that silica nanoparticles had a higher transfection efficiency than herpes simplex 1 vectors and showed less tissue damage. Mintzer and Simanek (2009) declares that "this success is a significant landmark in nonviral gene transfer, as such carriers typically exhibit low in vivo gene expression when compared to viral ana- logues".

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In recent years, carbon nanotubes have been used for delivering DNA and siRNA . A major hindrance for the use of carbon nanotubes as delivery vessels, is their insolubility. This has been addressed by oxidizing the tubes (Klumpp et al., 2006). Carbon nanotubes have been shown, in vitro, to successfully deliver DNA into cells via non-endocytotic routes (Pantarotto et al., 2004). Several studies have, both in vitro and in vivo, shown the potential of carbon nanotubes by successfully silencing genes by transfection of siRNA into T-cells, primary cells and dendritic cells (Kam et al., 2005; Liu et al., 2007b; Krajcik et al., 2008).

A slight shift in research focus can be observed. Recent studies have com- plemented the treatment efficacy of DNA delivery by magnetic homing mech- anisms. Ang et al. (2010) and Zhang et al. (2010) have, In vitro, successfully used iron oxide to assist gene delivery and increase efficiency of gene transfection. In vivo, Kievit et al. (2010) and Zhao et al. (2010) have been able to show that such targeted delivery systems are up to tenfold more efficient at increasing gene expression in mouse tumor models, compared to that of only nanoparticles.

1.4 Design of a nanoparticle cationic pH sensitive gene delivery vessel

Previous studies have concluded that an effective gene delivery vessel should be positively charged, preferably nano sized, stable outside the cell while allowing rapid internalization of DNA plasmids inside the cell nucleus, be biodegradable and have low toxicity. However, problems in previous systems include: low complex solubility (Klumpp et al., 2006) and unmodified-particle uptake by cells (Kim et al., 2010; Basarkar and Singh, 2009), high toxicity (Chollet et al., 2002; Moghimi et al., 2005) and low gene transfection (Lee et al., 2009; Huang et al., 2010). In this study we intend to address these key issues, by synthesizing nanoparticles, encapsulated with magnetic particles, using Eudragit polymers.

Evonik Industries have developed Eudragit polymers, which are pH-sensitive poly(methacrylic acid-co-methyl methacrylate) copolymers (Eudragit E PO, Eudragit L 100-55, Eudragit L 100, Eudragit S 100).

These polymers have been used previously in nanoparticulate formations, functioning as drug carriers (Jain et al., 2005; C. Bothiraja and Sher, 2009;

Devarajan and Sonavane, 2007; Dai et al., 2004). Eudragit polymers have also been FDA approved (FDA, 2010). The polymers are swellable at pH below 5, above 5.5, above 6.0 and above 7.0, respectively (Evonik-Industries, 2010). Previous studies have shown that Eudragit RS and RL nanoparticles can act as gene delivery vessels into different cell types with low toxicity and nanoparticle size independence (Gargouri et al., 2009; Cortesi et al., 2004; Wang WX, 2003). Eudragit E 100 polymer mixed with PLGA to form

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nanoparticles, has been shown to have significantly higher delivery of DNA into cells, compared to pure PLGA nanoparticles (Basarkar and Singh, 2009).

This effect is likely to be due to the differences in their processing in the endosome after uptake. The mix of polymers has a higher positive charge, that induces higher disruption capabilities of the endosomal wall.

Human Multi-potent Stromal Cells (or Mesenchymal Stem Cells) (MSCs) will be used in this study for testing our system. Kim et al. (2010) have previously revealed that human MSCs can successfully be used as model for gene transfection using PLGA and PLGA-Polyethylenimine (PEI) nanoparticles as carriers of SOX9 DNA complexes. Polyplexing with polyethylenimine (PEI) enhanced the cellular uptake of SOX9 DNA complexed with PLGA nanoparticles both in vitro and in vivo. These findings indicate that MSCs cell lines are suitable for gene transfection studies.

Here we develop a system with Eudragit E PO polymer nanoparticles, encapsulated with iron oxide particles, that act as gene therapy vessels by delivering DNA plasmids to the nucleus of cells. Iron oxide particles will aid in the translocation of the DNA-loaded particles to specific target cells, through the use of a magnetic fields. The iron oxide-DNA-loaded particles are easily taken up by cells through endocytotic pathways and quickly degraded inside the endosomes, releasing free DNA for relocation to, and processing inside, the cell nucleus. A conceptual outline of this system is given in Figure 1.1.

Specifically in this study, we will focus on particle size control as cellular internalization is affected by particle size, proof of concept of particle dissolution in low pH, particle surface charge analysis, cellular uptake and digestion of particles and toxicity study of the synthesized EPO particles. Later, we will study the efficacy of DNA plasmid delivery in vitro and in vivo, and also the effects of blending different ratios of Eudragit polymers with PLGA to maximize cellular uptake and minimize toxicity of the particles.

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Figure 1.1:Conceptual outline of the DNA delivery system. In the first step, magnetic iron oxide particles (black dots) are encapsulated inside EPO nanoparticles (large pur- ple circles). In the second step, DNA (green lines) is associated with the nanoparticles.

In the third step, these complexes are incubated with cells and taken up through endocytosis into endosomes (red ovals). Here, cellular uptake can be increased by using a magnetic field to attract the particles in a certain direction (red/black box represents a magnet). In the final step, the particles dissolve and disrupt the endosomes, releasing free DNA and iron oxide particles. The DNA is translocated into the nucleus (grey oval) and the iron oxide particles remain inside the cytoplasm.

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Part II:

Materials and Methods

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2. Materials and cell culturing

2.1 Materials

The pH-sensitive poly(methacrylic acid-co-methyl methacrylate) copolymers (Eudragit E PO, Eudragit L 100-55, Eudragit L 100, Eudragit S 100) (EPO, L100-55, L100, S100) were a generous gift from Röhm (Darmstadt, Ger- many). The Eudragit polymers were chosen for their pH sensitivity and previously known ability to form particles (Jain et al., 2005; C. Bothiraja and Sher, 2009; Devarajan and Sonavane, 2007; Dai et al., 2004). The polymers are swellable at pH below 5, above 5.5, above 6.0 and above 7.0, respectively. Millipore water was prepared by a Milli-Q Plus System (Millipore Corporation, Breford, USA). Universal pH buffers were prepared using Boric and Citric acid and Sodium phosphate and adjusted to correct pH using hydrochloric acid and sodium hydroxide (Carmody, 1961). Magnetic iron oxide (Fe₃O₄) particles , coated with oleic acid and dispersed in hexane , were bought from Ocean NanoTech (Springdale, Arkansas, USA). Unless other- wise stated, all other reagents were bought from Sigma-Aldrich (USA) and of analytical grade. The structural formulae of the pH sensitive polymer are given in Figure 2.1.

2.2 Cell lines and culturing

For the in vitro experiments human Multi-potent Stromal Cells (MSCs) (Cen- ter for Gene Therapy, Texas A & M University, Texas, USA) and RAW264.7 cells (mouse leukaemic monocyte macrophage cell line) were used (Amer- ican Type Culture Collection, USA). MSCs were cultured at 37^◦C in Mini- mum Essential Medium-Alpha (MEM-alpha) medium (1x, Invitrogen, USA)

(a) Eudragit E PO (b) Eudragit S 100 (c) Eudragit L 100 (d) Eudragit L 100-55 Figure 2.1:Structural formulae of the pH sensitive polymers.

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enriched with 15% (v/v) Fetal Bovine Serum (FBS) , 1% (v/v) L-Glutamine (L-Glu) and 1% (v/v) antibiotics. RAW264.7 cells were cultured at 37^◦C in Dulbeccos Modified Essential Medium (DMEM) (1x, Invitrogen, USA) enriched with 10% FBS, 1% L-Glu and 1% (v/v) antibiotics. To detach ad- herent MSCs for passaging or for experiments, cells were incubated in 1x Trypsin/ethylenediaminetetraacetic acid (EDTA) solution at 37^◦C for 3 min.

MSCs and RAW264.7 cells were kept at densities between 2,500 and 10,000 cells/cm².

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3. Initial particle preparation and characterization

3.1 Preparation of Rhodamine 6G-loaded particles

Rhodamine 6G-loaded particles were prepared by double-emulsion solvent evaporation technique. To examine the effects of different organic phases, polymer concentrations and emulsion stabilizer concentrations on the particle morphology, we employed modified versions of the methods described by Jain et al. (2005), Sahoo et al. (2002) and C. Bothiraja and Sher (2009). In a typical experiment, a 0.5 ml internal aqueous phase (IAP) of 5%, 10% or 15% w/v Polyvinyl alcohol (PVA) and 0.5 mg hydrophilic Rhodamine 6G dye was emulsified with internal organic phase (IOP) for 1 minute using an ultra- sonic liquid processor (20 W output power; Sonicator 3000; QSonica, LLC., Newtown, CT, USA). The temperature was maintained at 4^◦C using an ice bath. The IOP consisted of 25 mg (0.5% w/v) or 150 mg (3% w/v) of polymer in 5 ml of, either, a mixed solvent system of dichloromethane-to-ethanol-to- isopropyl (DCM-EtOH-ISP, D-E-I) alcohol in a ratio of 5:6:4, DCM, EtOH or Methanol (MeOH). The resulting emulsion was sonicated (20W, 1 min) and added drop by drop to an 25 ml external aqueous phase (EAP) of 1% w/v PVA solution made with, either Millipore water, or 0.04M Tris buffer (pH 9) . The aqueous PVA solution acts as an emulsion stabilizer. Emulsification was continued using a homogenizer (Tissue Master-125 Watt Lab Homogenizer;

Omni International, Kennesaw, GA, USA) at maximum speed for 5 minutes.

The resulting emulsion was stirred at room temperature overnight to allow the solvent to evaporate. The emulsion was then centrifuged at 6,000 rpm for 30 seconds and the supernatant was collected and the mix of particles were washed 3 times with Millipore water, or 0.04M Tris buffer (pH 9), by centrifugation at 14,000 rpm for 5 minutes. The particles were resuspended in Millipore water, or 0.04M Tris buffer (pH 9), and frozen at -80^◦C for minimum 3 hours and then lyophilized (Freeze Dryer 4.5; Labconco Corporation, Kansas City, Missouri, USA) for minimum 24 hours. The final products were stored at 4^◦C. The protocol was modified, as described below, for each polymer, to generate different batches of particles.

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Table 3.1: Synthesized Rhodamine 6G-loaded EPO particles centrifuged at 14,000 rpm for 5 min.

Batch ID Organic phase % polymer % PVA

EPO-1 D-E-I 3 10

Table 3.2: Synthesized Rhodamine 6G-loaded EPO particles centrifuged at 14,000 rpm for 20 min.

EPO-2 MeOH 0.5 5

EPO-3 MeOH 0.5 10

EPO-4 MeOH 0.5 15

EPO-5 MeOH 3 5

EPO-6 MeOH 3 10

EPO-7 MeOH 3 15

EPO-8 D-E-I 0.5 5

EPO-9 D-E-I 0.5 10

EPO-10 D-E-I 0.5 15

EPO-11 D-E-I 3 5

EPO-12 D-E-I 3 10

EPO-13 D-E-I 3 15

3.1.1 Eudragit E PO Rhodamine 6G-loaded particles

EPO particles were collected using the centrifugation described above and the following protocol (Table 3.1. This batch was designated as EPO-1.

Then, EPO nanoparticles were collected using longer centrifugation time at regular centrifugation speed. The first centrifugation step was 1 minute at 1,000 rpm and the second step was 14,000 rpm for 20 minutes. The batches were designated as EPO-2, 3, ..., 13 and the specific protocols are given in Table 3.2.

3.1.2 Eudragit S 100, L 100 and L 100-55 Rhodamine 6G-loaded particles

S100, L100 and L100-55 particles were prepared as a typical experiment using the following protocol (Table 3.3, and designated as S100-1, L100-1 and L100-55-1.

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Table 3.3: Synthesized Rhodamine 6G-loaded S100, L100 and L100-55 particles centrifuged at 14,000 rpm for 5 min.

S100-1 D-E-I 3 10

L100-1 D-E-I 3 10

L100-55-1 D-E-I 3 10

Table 3.4: Synthesized Rhodamine 6G-loaded PLGA particles.

PLGA-1 DCM 3 10

3.1.3 PLGA Rhodamine 6G-loaded particles

Synthesized Poly(lactic-co-glycolic acid) (PLGA) particles were used as control throughout this study. PLGA particles were synthesized as a typical experiment using the following protocol (Table 3.4, and designated as PLGA-1.

3.2 Characterization of Rhodamine 6G-loaded particles

The morphology of the Rhodamine 6G-loaded particles was analyzed using Scanning Electron Microscopy (SEM) (JEOL 6320). EPO particles were prepared in Tris buffer, L100, L100-55 and S100 particles in pH 4 buffer and PLGA particles in Millipore water. The size distribution of the particles was measured by means of Dynamic Light Scattering (Zetasizer Nano; Malvern Industries, Malvern Worcestershire WR14 1XZ, United Kingdom) and Coul- ter Counter (Multisizer 3 Coulter Counter; Beckman Coulter, Inc., Brea, CA, USA).

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4. Dissolution profiles and in vitro experiments

4.1 Dissolution profiles of Eudragit E PO and PLGA particles

EPO polymer particles were chosen as a model system to test the dissolution of the Rhodamine 6G-loaded particles. The EPO polymer dissolve in pH solutions below 5.0 (Evonik-Industries, 2010), and thus the manufactured particles were expected to swell and disrupt in buffer with pH below 5.0. To test the dissolution of EPO, freeze dried particles were suspended in Tris buffer (pH 9, 0.04M) at 3 mg/ml. 1.5 ml of the suspension was added to 5 cm long cylindrical filter tubes (Spectra/Por molecularporous membrane tubing; flat width: 25 mm, diameter: 16 mm, vol/length: 2.0 ml/cm, MWCO: 12-14,000;

Spectrum Laboratories, Inc., Rancho Dominguez, CA, USA). The tubes were sealed and immersed into 100 ml buffer solutions (pH 3.0, 3.5, 4.0, 4.5, 5.0 and 7.0). The tubes were incubated in beakers with magnetic stirrers, stirring at 35 rpm, for 48 hours. At each time point (every 20 minutes for 3 hours, then every 30 minutes until 6 hours and measurements at 24 and 48 hours), three 200µl samples were collected from each buffer solution and the fluorescence intensity of the collected samples was measured immediately (excitation wavelength 544 nm; emission wavelength 590 nm, BMG FLUOstar galaxy; MTX Lab Systems, Inc. Vienna, Virginia, USA). The volume of the systems was kept constant by adding 600µl of respective pH buffer to each solution, at each time point. The pH of the solutions was monitored and kept constant throughout the experiment. In this setup, undissolved particles could not escape the filter tubes, whilst the released hydrophilic Rhodamine 6G dye from dissolved particles would pass through the filter membrane and into the solution, from which samples were collected. The experiments for pH 4.0, 5.0 and 7.0 were repeated in triplicate and in duplicate for pH 3.0, 3.5 and 4.5 solutions.

The dissolution of PLGA particles was measured in a similar manner. How- ever, with some minor differences in the experimental setup. 28.3 mg of PLGA was suspended in 14.4 ml Millipore water and added, as previously described, to pH buffers (pH 4.0, 5.0, 5.5, 6.0 and 7.0). Measurements were made every 30 minutes for 7 hours and then at 22.5, 27, 100, 119 and 125 hours.

Each time 1 ml of solution was collected and the fluorescence intensity of the solution was measured immediately (excitaion wavelength 525 nm, emis- 26

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sion wavelength 555 nm; RF-5301PC, Shimadzu Scientific Instruments, inc., Columbia, Maryland, USA).

In order to represent the measured intensities in milligram (mg) , calibration measurements were made for EPO Rhodamine 6G-loaded particles. 0,546 mg of EPO particles was dissolved in 40 µl of EtOH, added to 1.460 ml of pH buffers (pH 3.0, 3.5, 4.0, 4.5, 5.0 and 7.0) and diluted into 11 different dilutions. The fluorescence intensity of each dilution, including measurements of only pH buffers, was measured immediately (excitation wavelength 544 nm;

emission wavelength 590 nm, BMG FLUOstar galaxy; MTX Lab Systems, Inc. Vienna, Virginia, USA). Thus, mapping a specific intensity to a known concentration of dissolved particles. Measurements were made in triplicate. A linear trend line was fitted to each pH measurement, using the overall average of the measured intensities for pure buffer as fixed 0 mg level. The combined average of the trend lines was used as formula to convert previously measured intensities into mg.

A calibration curve for the dissolution of PLGA particles was based on the dissolution profile of EPO. Dilutions of dissolved EPO particles were prepared similarly as described above in PBS buffer. Fluorescence intensities were measured immediately (excitaion wavelength 525 nm, emission wavelength 555 nm; RF-5301PC, Shimadzu Scientific Instrumens, inc., Columbia, Maryland, USA).

4.2 In vitro uptake of Rhodamine 6G-loaded Eudragit E PO particles

EPO particle uptake by cells, was examined by measuring the fluorescence intensity of particles taken up by cells, after incubating cells with Rhodamine 6G-loaded EPO particles. In triplicate, MSCs and RAW264.7 cells were seeded at 500,000 and 800,000 cells per T25 plate (VWR, USA) and incubated for 3 hours in particle concentrations of 0.02 mg/ml, 0.1 mg/ml and 0.5 mg/ml of EPO particles, 0.5 mg/ml PLGA particles and controls without any particles in 1.5 ml of MEM-alpha and DMEM media respectively.

After incubation, the cells were washed twice with PBS buffer, centrifuged at 1,000 rpm for 5 minutes, the pellet was dissolved in PBS buffer and the fluorescence intensity was measured (excitaion wavelength 525 nm, emission wavelength 555 nm; RF-5301PC, Shimadzu Scientific Instrumens, inc., Columbia, Maryland, USA).

To convert the measured intensities into mg; 0.1 and 0.3 mg of Rhodamine 6G-loaded EPO particles were dissolved in 0.1 ml EtOH, diluted in 9.9 ml PBS buffer and the fluorescence intensities of the resulting solutions were measured and recorded.

The uptake of Rhodamine 6G-loaded EPO and PLGA particles was also investigated through microscopy (Nikon Eclipse TE2000-U). MSCs and

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RAW264.7 cells were seeded at 1,500 cells/cm² and incubated at 37^◦C with different particles concentrations (0.005, 0.02, 0.05, 0.1, 0.5 and 1.0 mg/ml) for up to 6 hours. Every half an hour images were taken to study the cell viability and particle uptake by the cells.

4.3 In vitro digestion of Rhodamine 6G-loaded Eudragit E PO particles

To quantitatively determine the amount of EPO particles taken up and digested by cells, the fluorescence intensity of particles associated with the cells after incubation was measured. In triplicate, MSCs and RAW264.7 cells were seeded at 500,000 and 800,000 cells per T25 plate (VWR, USA) and incubated for 3 hours in particle concentrations of 0.1 mg/ml and 0.5 mg/ml of EPO particles, 0.5 mg/ml PLGA particles and controls without any particles in 1.5 ml of MEM-alpha and DMEM media respectively. After incubation, the cells were washed twice with PBS buffer and lysed in 1.5 ml PBS buffer by heating the cell suspension to 90^◦C for 7 minutes. The lysed cells and non-digested particles were collected by centrifugation at 14,000 rpm for 20 minutes at 4^◦C. The supernatant, containing released dye from digested particles, was collected, immediately frozen and the intensity of the solution was later measured (excitation wavelength 544 nm; emission wavelength 590 nm, BMG FLUOstar galaxy; MTX Lab Systems, Inc. Vienna, Virginia, USA).

A calibration curve, to relate measured fluorescence intensities to mg of dissolved particles, was made by dissolving a known amount of Rhodamine 6G-loaded EPO particles in a small amount of EtOH to dissolve the particles, then diluting the solution by adding different amounts of PBS buffer and measuring the fluorescence intensities at different particle concentrations (excitation wavelength 544 nm; emission wavelength 590 nm, BMG FLUOstar galaxy; MTX Lab Systems, Inc. Vienna, Virginia, USA).

4.4 Endosomal pH of MSCs and RAW264.7 cells

The endosomal pH of MSCs, RAW264.7 and Lipopolysaccharide (LPS) activated MSCs and RAW264.7 cells was measured by a standard ratiometric imaging technique (Holopainen et al., 2001; Jiang et al., 1990). RAW264.7 cells can be activated by adding LPS to the cell culture media (Bisht et al., 2007; Kong and Ge, 2008). MSCs were, as control, incubated with LPS. How- ever, they are not "activated" in the sense as referred to, when discussing ac- tivation of macrophage like cells. Lysotracker yellow/blue (Invitrogen, USA) was used as an endo- and lysosomal pH indicator (Diwu et al., 1999). The MSCs and RAW264.7 cells were seeded at 15,000 cells/cm² onto coverslips.

Four batches of cells were prepared: activated MSCs and RAW264.7 and non- 28

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activated MSCs and RAW264.7 cells. Cells were incubated for 4 hours. At time 0, LPS was added to one batch of RAW264.7 and MSCs respectively;

yielding a final concentration of 0.1 µg LPS / ml. After 1 hour, Rhodamine 6G-loaded EPO particles were added to make a final solution of 0.1 mg EPO particles / mg. After 2 hours, 20 µl of 1 mM lysotracker yellow/blue was added onto each coverslip. After 4 hours the cells were washed twice with PBS buffer and mounted in the microscope (Nikon Eclipse TE2000-U).

The cells were excited at 15 s intervals alternately at 340 and 380 nm, and the emission was recorded at 490 nm with a camera. An imaging program (Simple PCI) calculated and recorded the mean fluorescence ratio for numer- ous endosomes inside cells, selected by drawing an ellipse around its image on the screen of the computer. Subsequently, the fluorescence emission intensity ratios were calculated and transformed into pH values using calibration curves.

The calibration curves was made by measuring the emission at wavelengths 340 and 380 nm, excited at 490 nm, of different pH solutions and plotting the ratio of the measured values as a function of pH (RF-5301PC, Shimadzu Scientific Instrumens, inc., Columbia, Maryland, USA).

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5. Particle system using iron oxide-loaded nanoparticles

5.1 Preparation of iron oxide-loaded nanoparticles

Iron oxide-loaded and iron oxide- & C18 3,3’-dioctadecyloxacarbocyanine perchlorate (DiO)-loaded EPO and iron oxide-loaded PLGA nanoparticles were prepared by sonication-emulsification method (Ngaboni Okassa et al., 2005; Bilati et al., 2003). We employed a modified version of the method described by Liu et al. (2007a). Magnetic iron oxide particles in solution, coated with oleic acid, were precipitated out by washing with acetone. The iron oxide particles (24.9 mg in iron oxide-loaded EPO and 6.4 mg in iron oxide- & DiO-loaded EPO) were dried using nitrogen gas and dispersed into 2 ml DCM solution containing, only 100 mg polymer, or 100 mg polymer and 0.1 mg of the hydrophobic dye DiO. The solution was mixed by vortexing to form a stable oily suspension and added into 4 ml aqueous solution surfactant to help stabilize the emulsion (3% PVA). The emulsion was then sonicated (20W, 1 min) and added drop by drop into a 60 ml 1% w/v PVA solution stirred at high speed. The emulsion was stirred overnight to ensure complete evaporation of organic solvent. The emulsion was then centrifuged at 1,000 rpm for 10 minutes, the supernatant was collected and the nanoparticles in the supernatant were collected by centrifugation at 14,000 rpm for 10 minutes and washed 3 times with Millipore water. The particles were resuspended in Millipore water and frozen at -80^◦C for minimum 3 hours and then lyophilized for minimum 24 hours. The final products were stored at 4^◦C.

The synthesized iron oxide-loaded EPO and iron oxide- & DiO-loaded EPO were designated as EPO-I and EPO-D respectively.

5.2 Characterization of iron oxide-loaded particles

The morphology of the iron oxide-loaded nanoparticles was analyzed using Transmission Electron Microscope (TEM) (JEOL 200CX). The size distribution of the particles was measured by means of Dynamic Light Scattering and Coulter Counter. Zeta potential of nanoparticle suspensions was determined by Dynamic Light Scattering.

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5.3 In vitro uptake of iron oxide-loaded Eudragit E PO particles

Fluorescence-activated cell sorting (FACS) was applied to study the cellular uptake of iron oxide- & DiO-loaded EPO particles. Cells incubated with and without particles were compared. 1 mg of EPO particles were mixed with 300 µ l PBS and divided into 2 batches; with and without 500 µ l Poly-L-Lysine (PLL) solution added. PLL was added to particles, to investigate whether the positively charged PLL could aid in particle uptake by the cells. The particles were incubated for 2 hours, and then spun for 10 min at 6,000 rpm, resuspended in 1 ml PBS and spun again. Then, the particles were resuspended in 4 ml of MEM-alpha media with 3 different concentrations (6, 25 and 100 µ g/ml) and incubated with cells for 1 hour at 37^◦C. 500 µl solution of particles was saved for later analysis of pure particles. The cells were washed twice with PBS buffer and then trypsinzed and suspended in 200 µl of PBS buffer.

These solutions were run through in FACS.

5.4 Confocal microscopy of cells incubated with iron oxide & DiO-loaded Eudragit E PO particles

To study the uptake of iron oxide & DiO-loaded EPO particles in MSCs and RAW264.7, the cells were seeded at a density of 10,000 and 20,000 cells/well, respectively, onto a 24-well plate containing a cover slip. After incubation overnight the medium was replaced with fresh medium containing iron oxide- & DiO-loaded EPO particles (25 µg/ml). After incubating for 3 hours, the cells were washed twice with PBS buffer and the remaining cells were stained with 1,1’-Dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine perchlorate (DiD) dye for 5 minutes and then fixed in 3.7% formaldehyde for 20 minutes. The wells were then rinsed with PBS buffer twice. Finally, the cover slip in each well was transferred to glass slides, which were covered with mounting media (90% glycerol) . Cells were visualized and analyzed using a confocal microscope (Zeiss 510). This type protocol has also been applied by Jung et al. (2007).

5.5 Toxicity of iron oxide- & DiO-loaded Eudragit E PO particles in MSCs

The toxicity of iron oxide- & DiO-loaded EPO particles was measured using a mitochondrial activity (MTT assay; Invitrogen, USA). MSCs were plated with a density of 10,000 cells per well in a 96 well plate. After incubating overnight, the media was replaced with 100 µl fresh media and 10 µl of 12 mM MTT stock solution. Controls without MTT solution were also prepared.

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The cells were then incubated for 4 hours at 37^◦C and 100 µl Sodium Dodecyl Sulfate- (SDS)-HCl solution was added and cells were incubated for 4 hours again at 37^◦C. Absorbance measurements were made at wavelength 570 nm (BMG FLUOstar galaxy; MTX Lab Systems, Inc. Vienna, Virginia, USA).

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Part III:

Results

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6. Initial particle preparation and characterization

6.1 Characterization of Rhodamine 6G-loaded particles

6.1.1 Size distributions of Rhodamine 6G-loaded particles

Given in Table 6.1, are the synthesized Rhodamine 6G-loaded particles with their respective size distributions, given by DLS, if measured. Typical size distributions, as measured by Coulter Counter technology, are given in Fig- ure 6.4. DLS and Coulter Counter measurements were consistent within the batches, with following exceptions: EPO-3, EPO-4, EPO-9, EPO-11, EPO-12 and EPO-13. In these batches. 5% and 15 concentration in the IAP produced larger particles than 10 concentration, see Figure 6.1. Furthermore, a lower polymer concentration yields smaller particles when using MeOH as organic phase. At a concentration of 10% PVA in the IAP and 3% polymer in the IOP, there is no size difference between using D-E-I mix or MeOH as organic phase. In general, using MeOH as organic phase produces smaller particles.

See Figure 6.2. Longer centrifugation time decreases average diameter of the particles, as shown in Figure 6.3.

6.1.2 SEM of Eudragit polymer and PLGA particles

The shape and surface characteristics of Eudragit Rhodamine 6G-loaded nanoparticles are shown in Figure 6.5. There is a wide size distribution in all particle batches. The surfaces of the particles in batch S100-1 are not smooth.

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(a) D-E-I as organic phase. (b) MeOH as organic phase.

Figure 6.1:The effect of PVA concentration in the IAP, on average diameter of Rho- damine 6G-loaded particles. An IAP with 10 produces particles with the smallest diameter.

(a) 0.5% polymer. (b) 3 % polymer.

Figure 6.2:Effect of IOP on average diameter of Rhodamine 6G-loaded particles. A lower polymer concentration yields smaller particles, when using MeOH as organic phase. At a concentration of 10 in the IAP, there is no size difference between using D-E-I mix or MeOH as organic phase. In general, using MeOH as organic phase produces smaller particles.

A novel design of gene therapy carriers - pH sensitive cationic nanoparticles with encapsulated iron oxide particles