a novel design of gene therapy carriers - ph …...a novel design of gene therapy carriers - ph...

UPTEC X 010 27

Examensarbete 20 pNovember 2010

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

Jens Roat Kultima

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

M.SC. THESIS

A novel design of gene therapy carriers -pH sensitive cationic nanoparticles

with encapsulated iron oxide particles

JENS ROAT KULTIMA1−5, B.Sc.

SupervisorsJEFFREY M. KARP2−5, Ph.D.

CHENJIE XU2−5, Ph.D.

Scientific ReviewerJÖNS HILBORN1, Ph.D.

ExaminatorMARGARETA KRABBE1, Ph.D.

1Uppsala University2Center for Regenerative Therapeutics &

Department of Medicine, Brigham & Women’s Hospital3Harvard Medical School

4Harvard Stem Cell Institute5Harvard-MIT Division of Health Science and Technology

November 29, 2010



JENS ROAT KULTIMA, B.Sc.

AbstractGene 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 methodsof delivering these plasmids. Different types of vectors have been developed, withan initial focus on viral vectors. Due to cost, toxicity and adverse immune problemsof these vessels, focus has shifted more towards nonviral delivery systems andspecifically different types of nanoparticles with high biocompatibility and potentialfor large-scale production. There are several barriers a potential DNA carrier mustovercome: avoid clearance by the reticuloendothelial system, vessel delivery andadhesion to the cell surface, entry into the cell, endosomal escape and finally nucleartranslocation of conjugated DNA. Surface modification with hydrophilic polymers orplasma protein can prevent clearance form the reticuloendothelial system. Magnetismhas been proposed to control particle localization to organs, tissues and cells. It hasbeen shown that cellular uptake of DNA varies significantly based on carrier vector.Nanoparticles have been modified by adding cationic macromolecules onto thesurface of these particles to maximize the efficiency of gene transfection. Once thenanoparticles have reached the inside of the cell through endocytois, the endosomalwall has to be penetrated. This has been explored using several different methods;such as using chloroquine, a well-known lysosomotropic agent or the incoorperationof membrane-destabilizing peptides. Here we present a homing gene delivery carrier,consisting of cationic pH sensitive nanoparticles with encapsulated iron oxideparticles. The iron oxide particles enable the nanoparticles to be translocated to thecell surfaces through the use of an magnetic field. The cationic properties of thenanoparticle will enable cell surface adhesion. The pH sensitive nanoparticles willdegrade once inside the cellular endosomes and quickly disrupt the endosomal walls,allowing conjugated DNA to be translocated into the cell nucleus. Our system willincrease the uptake of particles, improve the translocation of gene vector from carrierparticles into cytoplasm, and reduce the cost of cell transfection.

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



JENS ROAT KULTIMA, B.Sc.

Populärvetenskaplig sammanfattningI slutet av åttio- och början av nittiotalet kom de första vetenskapliga

rapporterna som visar att det går att implantera funktionella varianter avdefekta gener i celler och få dessa celler att uttrycka det protein som kodasav den funktionella genen. Detta var inledningen till begreppet genterapi,som i korthet går ut på att transfektera nytt funktionellt genetisk materialtill cellen och därmed förändra cellens uttryck av protiner. För att kunnatransfektera celler effektivt måste dessa gener, eller DNA segment, effektivtföras in i målcellen. För att lösa det här problemet har forskare utvecklatolika metoder att inkapsla DNA i olika typer av bärare. En sådan typ avbärare kan vara nanopartiklar. Dessa nanopartiklar har tidigare bestått av t.ex.guld eller silica. De första metoderna för att föra in partiklarna i målcellernavar att skjuta in dem. Senare metoder använder sig av cellens egna systemför upptag, exempelvis genom endocytos. I det här arbetet presenterar vien bärare som består av pH känsliga nanopartiklar uppbyggda av en pHkänslig polymer med inkapslade järnoxidpartiklar, till vilka gener eller DNAfragment kan tillfogats genom adhesion. De syntetiserade nanopartiklarnaupptas lätt av cellen och väl inne i cellerna bryts de ner snabbt. Sedan frigörsinnehållet i partiklarna och detta kan sedan tas sig in i cellkärnan.

To mom and dad for supporting me in everything I do.To Elisabeth for always standing by my side.

Contents

List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viiList of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ixNomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiIndex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii

Part I: Introduction1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1.1 The birth of nanoparticle gene therapy . . . . . . . . . . . . . . . . . . . 141.2 In vitro barriers for cellular nanoparticle uptake . . . . . . . . . . . . 151.3 Previous uses of nanoparticles as gene carriers . . . . . . . . . . . . . 161.4 Design of a nanoparticle cationic pH sensitive gene delivery

vessel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Part II: Materials and Methods2 Materials and cell culturing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212.2 Cell lines and culturing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

3 Initial particle preparation and characterization . . . . . . . . . . . . . . . . 233.1 Preparation of Rhodamine 6G-loaded particles . . . . . . . . . . . . . 23

3.1.1 Eudragit E PO Rhodamine 6G-loaded particles . . . . . . . . 243.1.2 Eudragit S 100, L 100 and L 100-55 Rhodamine 6G-

loaded particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243.1.3 PLGA Rhodamine 6G-loaded particles . . . . . . . . . . . . . . . 25

3.2 Characterization of Rhodamine 6G-loaded particles . . . . . . . . . 254 Dissolution profiles and in vitro experiments . . . . . . . . . . . . . . . . . 26

4.1 Dissolution profiles of Eudragit E PO and PLGA particles . . . . 264.2 In vitro uptake of Rhodamine 6G-loaded Eudragit E PO particles 274.3 In vitro digestion of Rhodamine 6G-loaded Eudragit E PO par-

ticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284.4 Endosomal pH of MSCs and RAW264.7 cells . . . . . . . . . . . . . 28

5 Particle system using iron oxide-loaded nanoparticles . . . . . . . . . . . 305.1 Preparation of iron oxide-loaded nanoparticles . . . . . . . . . . . . . 305.2 Characterization of iron oxide-loaded particles . . . . . . . . . . . . . 305.3 In vitro uptake of iron oxide-loaded Eudragit E PO particles . . . 315.4 Confocal microscopy of cells incubated with iron oxide &

DiO-loaded Eudragit E PO particles . . . . . . . . . . . . . . . . . . . . . 31

5.5 Toxicity of iron oxide- & DiO-loaded Eudragit E PO particlesin MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Part III: Results6 Initial particle preparation and characterization . . . . . . . . . . . . . . . . 34

6.1 Characterization of Rhodamine 6G-loaded particles . . . . . . . . . 346.1.1 Size distributions of Rhodamine 6G-loaded particles . . . . 346.1.2 SEM of Eudragit polymer and PLGA particles . . . . . . . . . 34

7 Dissolution profiles and in vitro experiments . . . . . . . . . . . . . . . . . 397.1 Dissolution profiles of Eudragit E PO and PLGA particles . . . . 397.2 In vitro uptake of Rhodamine 6G-loaded Eudragit E PO particles 397.3 In vitro digestion of Rhodamine 6G-loaded Eudragit E PO par-

ticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407.4 Endosomal pH of MSCs and RAW264.7 cells . . . . . . . . . . . . . 407.5 Toxicity of Rhodamine 6G-loaded Eudragit E PO particles in

MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418 Particle system using iron oxide-loaded nanoparticles . . . . . . . . . . . 50

8.1 Characterization of iron oxide-loaded particles . . . . . . . . . . . . . 508.1.1 Size distributions of Rhodamine 6G-loaded particles . . . . 508.1.2 TEM images of iron oxide-loaded Eudragit E PO particles 508.1.3 Zeta potential of iron oxide-loaded EPO and PLGA particles 50

8.2 In vitro uptake of iron oxide- & DiO-loaded Eudragit E POparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

8.3 Confocal microscopy of cells incubated with iron oxide- &DiO-loaded Eudragit E PO particles . . . . . . . . . . . . . . . . . . . . . 51

8.4 Toxicity of iron oxide- & DiO-loaded Eudragit E PO particlesin MSCs and RAW264.7 cells . . . . . . . . . . . . . . . . . . . . . . . . . 51

Part IV: Discussion9 Initial particle preparation and characterization . . . . . . . . . . . . . . . . 57

9.1 Preparation of Rhodamine 6G-loaded particles . . . . . . . . . . . . . 579.2 Characterization of Rhodamine 6G-loaded particles . . . . . . . . . 57

10 Dissolution profiles and in vitro experiments . . . . . . . . . . . . . . . . . 5910.1 Dissolution profiles of Eudragit E PO and PLGA particles . . . . 5910.2 In vitro uptake and digestion of Rhodamine 6G-loaded Eu-

dragit E PO particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6010.3 Endosomal pH of MSCs and RAW264.7 cells . . . . . . . . . . . . . 6110.4 Toxicity of Rhodamine 6G-loaded Eudragit E PO particles in

MSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6111 Particle system using iron oxide-loaded nanoparticles . . . . . . . . . . . 62

11.1 Characterization of iron oxide-loaded particles . . . . . . . . . . . . . 6211.2 In vitro uptake of iron oxide- & DiO-loaded Eudragit E PO

particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

11.3 Confocal microscopy of cells incubated with iron oxide- &DiO-loaded Eudragit E PO particles . . . . . . . . . . . . . . . . . . . . . 63

11.4 Toxicity of iron oxide- & DiO-loaded Eudragit E PO particlesin MSCs and RAW264.7 cells . . . . . . . . . . . . . . . . . . . . . . . . . 63

Part V: Conclusions and Future Work12 Conclusions and Future Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Part VI: AcknowledgementsAcknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Part VII: BibliographyBibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

List of Tables

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

3.2 Synthesized Rhodamine 6G-loaded EPO particles centrifugedat 14,000 rpm for 20 min. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

3.3 Synthesized Rhodamine 6G-loaded S100, L100 and L100-55particles 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 theirrespective size distributions, given by DLS and CoulterCounter (CC), if measured. . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

7.1 Endosomal pH of non-activated and LPS activated MSCs andRAW264.7 cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

8.1 Synthesized iron oxide- and iron oxide &DiO-loaded particlesand their respective size distributions, given by DLS. . . . . . . . 50

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-loadedparticles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

6.3 Effect of centrifugation time on average diameter ofRhodamine 6G-loaded particles. . . . . . . . . . . . . . . . . . . . . . . . 36

6.4 Typical size distribution of Rhodamine 6G-loaded EPO particles 376.5 Representative SEM images of Rhodamine 6G-loaded EPO,

S100 and PLGA particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

7.1 Dissolution profiles of Rhodamine 6G-loaded EPO particlesin different pH buffers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

7.2 Dissolution profiles of Rhodamine 6G-loaded PLGA particlesin different pH buffers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

7.3 Rhodamine 6G-loaded EPO particles, after 6 hours, incubatedin 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 particlesfor MSCs and RAW264.7 cells. . . . . . . . . . . . . . . . . . . . . . . . 44

7.7 Microsopic images of MSCs incubated with 0.5, 0.1 and 0.02mg EPO particles / ml. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

7.8 Microsopic images of RAW264.7 cells incubated with 0.5, 0.1and 0.02 mg EPO particles / ml. . . . . . . . . . . . . . . . . . . . . . . . 46

7.9 Digestion efficiency of Rhodamine 6G-loaded EPO andPLGA particles for MSCs and RAW264.7 cells. . . . . . . . . . . . 47

7.10 Calibration curve for the dissolution profiles of Rhodamine6G-loaded EPO and PLGA particles for the in vitro digestionexperiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

7.11 Calibration curves used to calculate pH values from measuredfluorescence intensities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

7.12 Microscopic images of RAW264.7 cells and MSCs incubatedwith and without LPS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

7.13 Microscopic images of MSCs incubated with different con-centrations of Rhodamine 6G-loaded EPO particles at differ-ent 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-loadedEPO particles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

8.2 Zeta potential of iron oxide-loaded EPO and PLGA particles. . 538.3 FACS results of MSCs incubated without particles, with EPO

particles and also with EPO-PLL particles. . . . . . . . . . . . . . . . 538.4 Confocal microscopic images of MSCs and RAW264.7 cells

incubated with iron oxide- & DiO-loaded EPO particles. . . . . 548.5 Cell viability of MSCs and RAW264.7 cells, after incubating

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

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’-tetramethylindodicarbocyanineperchlorate

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

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

Index

Boric acid, 19

Citric acid, 19Coulter Counter, 23

D-E-I, 21Dichloromethane, 21DiD, 29DiO, 28DMEM, 20double-emulsion technique, 21Dynamic Light Scattering, 23

EDTA, 20Ethanol, 21Eudragit E PO, 19Eudragit L 100, 19Eudragit L 100-55, 19Eudragit S 100, 19external aqueous phase, 21

Fe3O4, 19Fetal Bovine Serum, 19

glycerol, 29

hexane, 19Hydrochloric acid, 19

internal aqueous phase, 21internal organic phase, 21iron oxide particles, 19

L-Glutamine, 19Lipopolysaccharide, 26Lysotracker yellow/blue, 26

MEM-alpha, 19Mesenchymal Stem Cells, 19

Methanol, 21Multi-potent Stromal Cells, 19

Oleic acid, 19

pH sensitivity, 19PLGA, 23Poly-L-Lysine, 29Polyethylenimine, 16Polyvinyl alcohol, 21

RAW264.7 cells, 19Rhodamine 6G, 21

SDS-HCl solution, 30Simple PCI, 27siRNA, 15Sodium hydroxide, 19Sodium phosphate, 19sonication-emulsification method, 28

Tris buffer, 21Trypsin, 20

Part I:

Introduction

1. Introduction

1.1 The birth of nanoparticle gene therapyIn the 80’s and 90’s, it was shown that diseases caused by a specific geneticdefect 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 interestedover the past decade (Gabhann et al., 2010; Duceppe and Tabrizian, 2010).Research has mainly been focused on developing nucleic acids (DNA) to bedelivered and methods of delivering the DNA (Patil et al., 2005).

Different types of vectors have been developed and employed for gene de-livery; bacterial vectors (Darji et al., 1997; Loessner and Weiss, 2004), vi-ral 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 genetherapy; severe combined immunodeficiency (Cavazzana-Calvo et al., 2000)and Parkinson′s disease (Kaplitt et al., 2007). Also tissue engineering, specif-ically using Multi-potent Stromal Cells (MSCs), has been a target for genetherapy (Goessler et al., 2006). The use of viral vectors in clinical applications,as gene delivery vehicles, has several limitations: toxicity, high cost, limitedquality, cause of cell damage, and viral vectors can also induce adverse im-mune problems (Verma and Somia, 1997; Bergen et al., 2008; Mörner et al.,2009). Due to the limitations of viral vectors, research has focused more onother types of delivery vessels, such as polypeptides and nanoparticles. Thesecompounds have high biocompatibility and potential for large-scale produc-tion (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 bloodvessels to reach the target cells. Simultaneously, the particles have to passthrough the reticuloendothelial system to avoid the clearance by B or T cells.Once the particles reach the target cells, the most important steps are entryinto the cell cytoplasm and release of DNA. Nanoparticles, with encapsulatedDNA or DNA conjugated into the particle surface, can enter cells by endocyto-sis or phagocytosis. DNA can also be encapsulated inside particles and phys-ically shot into cells through electric charge. (Uchimura et al., 2007; Hauck

14

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

1.2 In vitro barriers for cellular nanoparticle uptakeTo achieve successful treatment of a disease using gene therapy, the therapeu-tic DNA must be delivered into the target cells. The DNA can be deliveredwith or without the use of a delivery vessel. Cellular uptake of free DNA viaplasma membrane permeation is hindered by the size and negative charge ofthe DNA molecule and systemic circulation of DNA is hindered by nucleasedegradation (Mintzer and Simanek, 2009). It has been shown that cellular up-take of DNA varies significantly based on carrier vector, cell type and surfaceglycosaminoglycans (GAGs) present on the cell and that the internalized com-plexes may be delivered into intracellular compartments that do not promotetranscription (Ruponen et al., 2004). Nanoparticles have been modified byadding cationic macromolecules onto the nanoparticles to optimize deliveryof genes (Andersen et al., 2010), by increasing the electrostatic differencesbetween particle and cell surfaces. Ionic-ionic complexes formed by nega-tively charged DNA and positively charged nanoparticles have been used inboth in vitro and in vivo studies to increase transfection of intact DNA (Zenget al., 2010).

Cellular uptake of cationic complexes has been shown to proceed throughdifferent endocytic routes, such as clathrin-dependent endocytosis or bymacropinocytosis, in different cell types (Kopatz et al., 2004; Gonçalveset al., 2004). The morphology of DNA complex formed with cationicpolymers is independent of the polymer used, i.e. different cationic polymersinteract similarly with the DNA to form complexes with similar surfaceproperties and comparable uptake efficiencies. Though, the size of theformed nanoparticles effect the uptake efficiency (Mintzer and Simanek,2009; Desai et al., 1997; Prabha et al., 2002). The optimal size of complexesfor highest uptake is a mean diameter less than 200 nm. However, "enforced"endocytosis may be promoted by changing the surface charge and thusallowing larger complexes with more surface area be internalized by the cell(Peer et al., 2007; Mintzer and Simanek, 2009; Aoyama et al., 2003). Thetheoretical optimal mean diameter of carriers for cellular uptake has beencalculated to 54-60 nm (Gao et al., 2005).

Once the nanoparticles have reached the inside of the cell through endo-cytosis, they are located in lysosomes or endosomes. These compartmentsare 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 thecell cytoplasm, the endosomal wall have to be penetrated. This has been ex-plored using several different methods; such as using chloroquine, a well-

15

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

Complexes less than 9-11 nm in diameter can passively enter the nucleusthrough pores in the nuclear membrane (Bonner, 1975), and larger structuresenter the nucleus in an ATP-dependent process. This process is triggered byrecognition of short peptide sequences and hindered by certain antinucleo-porin antibodies (Featherstone et al., 1988). Plasmid DNA can enter the nu-cleus more easily during cell division and positively charged vectors promotenuclear-localizing effect (Wilke et al., 1996; Pouton and Seymour, 2001).Mintzer and Simanek (2009) concludes that "regardless of the exact method ofnuclear entry, gene sequences complexed with cationic vector systems seemto have an advantage over free plasmid DNA for in vitro cell transfection".

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

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

16

In recent years, carbon nanotubes have been used for delivering DNAand siRNA . A major hindrance for the use of carbon nanotubes as deliveryvessels, is their insolubility. This has been addressed by oxidizing the tubes(Klumpp et al., 2006). Carbon nanotubes have been shown, in vitro, tosuccessfully deliver DNA into cells via non-endocytotic routes (Pantarottoet al., 2004). Several studies have, both in vitro and in vivo, shown thepotential of carbon nanotubes by successfully silencing genes by transfectionof siRNA into T-cells, primary cells and dendritic cells (Kam et al., 2005; Liuet 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, successfullyused iron oxide to assist gene delivery and increase efficiency of gene trans-fection. In vivo, Kievit et al. (2010) and Zhao et al. (2010) have been able toshow that such targeted delivery systems are up to tenfold more efficient atincreasing gene expression in mouse tumor models, compared to that of onlynanoparticles.

1.4 Design of a nanoparticle cationic pH sensitive genedelivery vesselPrevious studies have concluded that an effective gene delivery vessel shouldbe positively charged, preferably nano sized, stable outside the cell whileallowing rapid internalization of DNA plasmids inside the cell nucleus, bebiodegradable and have low toxicity. However, problems in previous systemsinclude: low complex solubility (Klumpp et al., 2006) and unmodified-particleuptake by cells (Kim et al., 2010; Basarkar and Singh, 2009), high toxicity(Chollet et al., 2002; Moghimi et al., 2005) and low gene transfection (Leeet al., 2009; Huang et al., 2010). In this study we intend to address these keyissues, by synthesizing nanoparticles, encapsulated with magnetic particles,using Eudragit polymers.

Evonik Industries have developed Eudragit polymers, which arepH-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 havealso been FDA approved (FDA, 2010). The polymers are swellable at pHbelow 5, above 5.5, above 6.0 and above 7.0, respectively (Evonik-Industries,2010). Previous studies have shown that Eudragit RS and RL nanoparticlescan act as gene delivery vessels into different cell types with low toxicityand nanoparticle size independence (Gargouri et al., 2009; Cortesi et al.,2004; Wang WX, 2003). Eudragit E 100 polymer mixed with PLGA to form

17

nanoparticles, has been shown to have significantly higher delivery of DNAinto cells, compared to pure PLGA nanoparticles (Basarkar and Singh, 2009).This effect is likely to be due to the differences in their processing in theendosome 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 previ-ously revealed that human MSCs can successfully be used as model for genetransfection using PLGA and PLGA-Polyethylenimine (PEI) nanoparticles ascarriers of SOX9 DNA complexes. Polyplexing with polyethylenimine (PEI)enhanced the cellular uptake of SOX9 DNA complexed with PLGA nanopar-ticles both in vitro and in vivo. These findings indicate that MSCs cell linesare suitable for gene transfection studies.

Here we develop a system with Eudragit E PO polymer nanoparticles, en-capsulated with iron oxide particles, that act as gene therapy vessels by de-livering DNA plasmids to the nucleus of cells. Iron oxide particles will aid inthe translocation of the DNA-loaded particles to specific target cells, throughthe use of a magnetic fields. The iron oxide-DNA-loaded particles are easilytaken up by cells through endocytotic pathways and quickly degraded insidethe 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 cellularinternalization is affected by particle size, proof of concept of particle dissolu-tion in low pH, particle surface charge analysis, cellular uptake and digestionof particles and toxicity study of the synthesized EPO particles. Later, we willstudy the efficacy of DNA plasmid delivery in vitro and in vivo, and also theeffects of blending different ratios of Eudragit polymers with PLGA to maxi-mize cellular uptake and minimize toxicity of the particles.

18

Figure 1.1: Conceptual outline of the DNA delivery system. In the first step, magneticiron 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 endo-cytosis into endosomes (red ovals). Here, cellular uptake can be increased by using amagnetic field to attract the particles in a certain direction (red/black box represents amagnet). In the final step, the particles dissolve and disrupt the endosomes, releasingfree DNA and iron oxide particles. The DNA is translocated into the nucleus (greyoval) and the iron oxide particles remain inside the cytoplasm.

19

Part II:

Materials and Methods

2. Materials and cell culturing

2.1 MaterialsThe 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 pre-viously known ability to form particles (Jain et al., 2005; C. Bothiraja andSher, 2009; Devarajan and Sonavane, 2007; Dai et al., 2004). The polymersare swellable at pH below 5, above 5.5, above 6.0 and above 7.0, respec-tively. Millipore water was prepared by a Milli-Q Plus System (MilliporeCorporation, Breford, USA). Universal pH buffers were prepared using Boricand Citric acid and Sodium phosphate and adjusted to correct pH using hy-drochloric acid and sodium hydroxide (Carmody, 1961). Magnetic iron ox-ide (Fe3O4) particles , coated with oleic acid and dispersed in hexane , werebought from Ocean NanoTech (Springdale, Arkansas, USA). Unless other-wise stated, all other reagents were bought from Sigma-Aldrich (USA) andof analytical grade. The structural formulae of the pH sensitive polymer aregiven in Figure 2.1.

2.2 Cell lines and culturingFor the in vitro experiments human Multi-potent Stromal Cells (MSCs) (Cen-ter for Gene Therapy, Texas A & M University, Texas, USA) and RAW264.7cells (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.

21

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◦Cin 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 1xTrypsin/ethylenediaminetetraacetic acid (EDTA) solution at 37◦C for 3 min.MSCs and RAW264.7 cells were kept at densities between 2,500 and 10,000cells/cm2.

22

3. Initial particle preparation andcharacterization

3.1 Preparation of Rhodamine 6G-loaded particlesRhodamine 6G-loaded particles were prepared by double-emulsion solventevaporation technique. To examine the effects of different organic phases,polymer concentrations and emulsion stabilizer concentrations on the parti-cle morphology, we employed modified versions of the methods described byJain et al. (2005), Sahoo et al. (2002) and C. Bothiraja and Sher (2009). Ina typical experiment, a 0.5 ml internal aqueous phase (IAP) of 5%, 10% or15% w/v Polyvinyl alcohol (PVA) and 0.5 mg hydrophilic Rhodamine 6G dyewas 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 icebath. The IOP consisted of 25 mg (0.5% w/v) or 150 mg (3% w/v) of polymerin 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, EtOHor 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/vPVA solution made with, either Millipore water, or 0.04M Tris buffer (pH 9) .The aqueous PVA solution acts as an emulsion stabilizer. Emulsification wascontinued 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 allowthe solvent to evaporate. The emulsion was then centrifuged at 6,000 rpm for30 seconds and the supernatant was collected and the mix of particles werewashed 3 times with Millipore water, or 0.04M Tris buffer (pH 9), by cen-trifugation at 14,000 rpm for 5 minutes. The particles were resuspended inMillipore water, or 0.04M Tris buffer (pH 9), and frozen at -80◦C for mini-mum 3 hours and then lyophilized (Freeze Dryer 4.5; Labconco Corporation,Kansas City, Missouri, USA) for minimum 24 hours. The final products werestored at 4◦C. The protocol was modified, as described below, for each poly-mer, to generate different batches of particles.

23

Table 3.1: Synthesized Rhodamine 6G-loaded EPO particles centrifuged at 14,000rpm 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 14,000rpm for 20 min.


EPO-2 MeOH 0.5 5EPO-3 MeOH 0.5 10EPO-4 MeOH 0.5 15EPO-5 MeOH 3 5EPO-6 MeOH 3 10EPO-7 MeOH 3 15EPO-8 D-E-I 0.5 5EPO-9 D-E-I 0.5 10EPO-10 D-E-I 0.5 15EPO-11 D-E-I 3 5EPO-12 D-E-I 3 10EPO-13 D-E-I 3 15

3.1.1 Eudragit E PO Rhodamine 6G-loaded particlesEPO particles were collected using the centrifugation described above and thefollowing protocol (Table 3.1. This batch was designated as EPO-1.

Then, EPO nanoparticles were collected using longer centrifugation timeat regular centrifugation speed. The first centrifugation step was 1 minute at1,000 rpm and the second step was 14,000 rpm for 20 minutes. The batcheswere designated as EPO-2, 3, ..., 13 and the specific protocols are given inTable 3.2.

3.1.2 Eudragit S 100, L 100 and L 100-55 Rhodamine6G-loaded particlesS100, L100 and L100-55 particles were prepared as a typical experiment us-ing the following protocol (Table 3.3, and designated as S100-1, L100-1 andL100-55-1.

24

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


S100-1 D-E-I 3 10L100-1 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 particlesSynthesized Poly(lactic-co-glycolic acid) (PLGA) particles were used as con-trol throughout this study. PLGA particles were synthesized as a typical ex-periment using the following protocol (Table 3.4, and designated as PLGA-1.

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

25

4. Dissolution profiles and in vitroexperiments

4.1 Dissolution profiles of Eudragit E PO and PLGAparticlesEPO polymer particles were chosen as a model system to test the dissolu-tion of the Rhodamine 6G-loaded particles. The EPO polymer dissolve in pHsolutions below 5.0 (Evonik-Industries, 2010), and thus the manufactured par-ticles were expected to swell and disrupt in buffer with pH below 5.0. To testthe 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 longcylindrical filter tubes (Spectra/Por molecularporous membrane tubing; flatwidth: 25 mm, diameter: 16 mm, vol/length: 2.0 ml/cm, MWCO: 12-14,000;Spectrum Laboratories, Inc., Rancho Dominguez, CA, USA). The tubes weresealed and immersed into 100 ml buffer solutions (pH 3.0, 3.5, 4.0, 4.5, 5.0and 7.0). The tubes were incubated in beakers with magnetic stirrers, stirringat 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 fluo-rescence intensity of the collected samples was measured immediately (ex-citation wavelength 544 nm; emission wavelength 590 nm, BMG FLUOstargalaxy; MTX Lab Systems, Inc. Vienna, Virginia, USA). The volume of thesystems was kept constant by adding 600µl of respective pH buffer to eachsolution, at each time point. The pH of the solutions was monitored and keptconstant throughout the experiment. In this setup, undissolved particles couldnot escape the filter tubes, whilst the released hydrophilic Rhodamine 6G dyefrom dissolved particles would pass through the filter membrane and into thesolution, from which samples were collected. The experiments for pH 4.0, 5.0and 7.0 were repeated in triplicate and in duplicate for pH 3.0, 3.5 and 4.5solutions.

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 PLGAwas 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 ev-ery 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 ofthe solution was measured immediately (excitaion wavelength 525 nm, emis-

26

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

In order to represent the measured intensities in milligram (mg) , calibrationmeasurements were made for EPO Rhodamine 6G-loaded particles. 0,546 mgof EPO particles was dissolved in 40 µl of EtOH, added to 1.460 ml of pHbuffers (pH 3.0, 3.5, 4.0, 4.5, 5.0 and 7.0) and diluted into 11 different dilu-tions. The fluorescence intensity of each dilution, including measurements ofonly 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 knownconcentration of dissolved particles. Measurements were made in triplicate. Alinear trend line was fitted to each pH measurement, using the overall averageof the measured intensities for pure buffer as fixed 0 mg level. The combinedaverage of the trend lines was used as formula to convert previously measuredintensities into mg.

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

4.2 In vitro uptake of Rhodamine 6G-loaded EudragitE PO particlesEPO particle uptake by cells, was examined by measuring the fluorescenceintensity of particles taken up by cells, after incubating cells with Rhodamine6G-loaded EPO particles. In triplicate, MSCs and RAW264.7 cells wereseeded at 500,000 and 800,000 cells per T25 plate (VWR, USA) andincubated for 3 hours in particle concentrations of 0.02 mg/ml, 0.1 mg/ml and0.5 mg/ml of EPO particles, 0.5 mg/ml PLGA particles and controls withoutany particles in 1.5 ml of MEM-alpha and DMEM media respectively.After incubation, the cells were washed twice with PBS buffer, centrifugedat 1,000 rpm for 5 minutes, the pellet was dissolved in PBS buffer and thefluorescence intensity was measured (excitaion wavelength 525 nm, emissionwavelength 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 Rhodamine6G-loaded EPO particles were dissolved in 0.1 ml EtOH, diluted in 9.9 mlPBS buffer and the fluorescence intensities of the resulting solutions weremeasured and recorded.

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

27

RAW264.7 cells were seeded at 1,500 cells/cm2 and incubated at 37◦C withdifferent 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 cellviability and particle uptake by the cells.

4.3 In vitro digestion of Rhodamine 6G-loadedEudragit E PO particlesTo quantitatively determine the amount of EPO particles taken up and di-gested by cells, the fluorescence intensity of particles associated with the cellsafter incubation was measured. In triplicate, MSCs and RAW264.7 cells wereseeded at 500,000 and 800,000 cells per T25 plate (VWR, USA) and incu-bated for 3 hours in particle concentrations of 0.1 mg/ml and 0.5 mg/ml ofEPO particles, 0.5 mg/ml PLGA particles and controls without any particlesin 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 bufferby heating the cell suspension to 90◦C for 7 minutes. The lysed cells andnon-digested particles were collected by centrifugation at 14,000 rpm for 20minutes at 4◦C. The supernatant, containing released dye from digested parti-cles, was collected, immediately frozen and the intensity of the solution waslater 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 ofdissolved particles, was made by dissolving a known amount of Rhodamine6G-loaded EPO particles in a small amount of EtOH to dissolve the parti-cles, then diluting the solution by adding different amounts of PBS buffer andmeasuring the fluorescence intensities at different particle concentrations (ex-citation wavelength 544 nm; emission wavelength 590 nm, BMG FLUOstargalaxy; MTX Lab Systems, Inc. Vienna, Virginia, USA).

4.4 Endosomal pH of MSCs and RAW264.7 cellsThe endosomal pH of MSCs, RAW264.7 and Lipopolysaccharide (LPS) ac-tivated MSCs and RAW264.7 cells was measured by a standard ratiometricimaging technique (Holopainen et al., 2001; Jiang et al., 1990). RAW264.7cells 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). TheMSCs 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

activated MSCs and RAW264.7 cells. Cells were incubated for 4 hours. Attime 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, Rhodamine6G-loaded EPO particles were added to make a final solution of 0.1 mg EPOparticles / mg. After 2 hours, 20 µl of 1 mM lysotracker yellow/blue wasadded onto each coverslip. After 4 hours the cells were washed twice withPBS buffer and mounted in the microscope (Nikon Eclipse TE2000-U).

The cells were excited at 15 s intervals alternately at 340 and 380 nm, andthe 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 imageon the screen of the computer. Subsequently, the fluorescence emission inten-sity ratios were calculated and transformed into pH values using calibrationcurves.

The calibration curves was made by measuring the emission at wavelengths340 and 380 nm, excited at 490 nm, of different pH solutions and plottingthe ratio of the measured values as a function of pH (RF-5301PC, ShimadzuScientific Instrumens, inc., Columbia, Maryland, USA).

29

5. Particle system using ironoxide-loaded nanoparticles

5.1 Preparation of iron oxide-loaded nanoparticlesIron oxide-loaded and iron oxide- & C18 3,3’-dioctadecyloxacarbocyanineperchlorate (DiO)-loaded EPO and iron oxide-loaded PLGA nanoparticleswere prepared by sonication-emulsification method (Ngaboni Okassa et al.,2005; Bilati et al., 2003). We employed a modified version of the methoddescribed by Liu et al. (2007a). Magnetic iron oxide particles in solution,coated with oleic acid, were precipitated out by washing with acetone. Theiron oxide particles (24.9 mg in iron oxide-loaded EPO and 6.4 mg in ironoxide- & DiO-loaded EPO) were dried using nitrogen gas and dispersed into2 ml DCM solution containing, only 100 mg polymer, or 100 mg polymer and0.1 mg of the hydrophobic dye DiO. The solution was mixed by vortexing toform a stable oily suspension and added into 4 ml aqueous solution surfactantto 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 solutionstirred at high speed. The emulsion was stirred overnight to ensure completeevaporation of organic solvent. The emulsion was then centrifuged at 1,000rpm for 10 minutes, the supernatant was collected and the nanoparticles inthe supernatant were collected by centrifugation at 14,000 rpm for 10 minutesand washed 3 times with Millipore water. The particles were resuspended inMillipore water and frozen at -80◦C for minimum 3 hours and then lyophilizedfor minimum 24 hours. The final products were stored at 4◦C.

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

5.2 Characterization of iron oxide-loaded particlesThe morphology of the iron oxide-loaded nanoparticles was analyzed usingTransmission Electron Microscope (TEM) (JEOL 200CX). The size distribu-tion of the particles was measured by means of Dynamic Light Scattering andCoulter Counter. Zeta potential of nanoparticle suspensions was determinedby Dynamic Light Scattering.

30

5.3 In vitro uptake of iron oxide-loaded Eudragit E POparticlesFluorescence-activated cell sorting (FACS) was applied to study the cellularuptake of iron oxide- & DiO-loaded EPO particles. Cells incubated with andwithout particles were compared. 1 mg of EPO particles were mixed with 300µl PBS and divided into 2 batches; with and without 500 µl Poly-L-Lysine(PLL) solution added. PLL was added to particles, to investigate whether thepositively charged PLL could aid in particle uptake by the cells. The particleswere incubated for 2 hours, and then spun for 10 min at 6,000 rpm, resus-pended in 1 ml PBS and spun again. Then, the particles were resuspendedin 4 ml of MEM-alpha media with 3 different concentrations (6, 25 and 100µg/ml) and incubated with cells for 1 hour at 37◦C. 500 µl solution of parti-cles was saved for later analysis of pure particles. The cells were washed twicewith 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 ironoxide & DiO-loaded Eudragit E PO particlesTo study the uptake of iron oxide & DiO-loaded EPO particles in MSCsand RAW264.7, the cells were seeded at a density of 10,000 and 20,000cells/well, respectively, onto a 24-well plate containing a cover slip. Afterincubation overnight the medium was replaced with fresh medium containingiron oxide- & DiO-loaded EPO particles (25 µg/ml). After incubating for 3hours, the cells were washed twice with PBS buffer and the remaining cellswere stained with 1,1’-Dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanineperchlorate (DiD) dye for 5 minutes and then fixed in 3.7% formaldehydefor 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 coveredwith mounting media (90% glycerol) . Cells were visualized and analyzedusing a confocal microscope (Zeiss 510). This type protocol has also beenapplied by Jung et al. (2007).

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

31

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

32

Part III:

Results


6.1 Characterization of Rhodamine 6G-loadedparticles6.1.1 Size distributions of Rhodamine 6G-loaded particlesGiven in Table 6.1, are the synthesized Rhodamine 6G-loaded particles withtheir respective size distributions, given by DLS, if measured. Typical sizedistributions, as measured by Coulter Counter technology, are given in Fig-ure 6.4. DLS and Coulter Counter measurements were consistent within thebatches, with following exceptions: EPO-3, EPO-4, EPO-9, EPO-11, EPO-12and EPO-13. In these batches. 5% and 15 concentration in the IAP producedlarger particles than 10 concentration, see Figure 6.1. Furthermore, a lowerpolymer concentration yields smaller particles when using MeOH as organicphase. At a concentration of 10% PVA in the IAP and 3% polymer in theIOP, there is no size difference between using D-E-I mix or MeOH as organicphase. In general, using MeOH as organic phase produces smaller particles.See Figure 6.2. Longer centrifugation time decreases average diameter of theparticles, as shown in Figure 6.3.

6.1.2 SEM of Eudragit polymer and PLGA particlesThe shape and surface characteristics of Eudragit Rhodamine 6G-loadednanoparticles are shown in Figure 6.5. There is a wide size distribution in allparticle batches. The surfaces of the particles in batch S100-1 are not smooth.

34

(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 smallestdiameter.

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

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

35

Figure 6.3: Effect of centrifugation time on average diameter of Rhodamine 6G-loaded particles. Centrifuging for 20 minutes decreased the average diameter of theparticles from 2366 nm to 557 nm compared to 5 minutes centrifugation time.

Table 6.1: Synthesized Rhodamine 6G-loaded particles and their respective size dis-tributions, given by DLS and Coulter Counter (CC), if measured.

Batch ID IOP % Polymer % PVA DLS (nm) CC (nm)

EPO-1 D-E-I 3 10 2366 ± 430 N/Ab)

EPO-2 MeOH 0.5 5 587 ± 110 771 ± 652EPO-3 MeOH 0.5 10 199 ± 4 1053 ± 616EPO-4 MeOH 0.5 15 219 ± 12 765 ± 521EPO-5 MeOH 3 5 1325 ± 244 1018 ± 230EPO-6 MeOH 3 10 511 ± 51 744 ± 166EPO-7 MeOH 3 15 534 ± 61 674 ± 115EPO-8 D-E-I 0.5 5 2720 ± 1495 N/Aa)

EPO-9 D-E-I 0.5 10 1035 ± 95 679 ± 195EPO-10 D-E-I 0.5 15 N/Aa) 765 ± 229EPO-11 D-E-I 3 5 869 ± 19 1760 ± 1131EPO-12 D-E-I 3 10 557 ± 68 1578 ± 557EPO-13 D-E-I 3 15 2092 ± 44 1064 ± 227S100-1 D-E-I 3 10 3462 ± 653 N/Ab)

L100-1 D-E-I 3 10 3201 ± 903 N/Ab)

L100-55-1 D-E-I 3 10 5660 ± 871 N/Ab)

PLGA-1 DCM 3 10 707 ± 45 N/Ab)

a) These samples could not be measured due to experimental error. b) These samples were notmeasured.

36

Figure 6.4: Typical size distribution of Rhodamine 6G-loaded EPO particles madewith D-E-I mix as organic phases and measured by a Coulter Counter. The distribu-tion, for this batch, correlates well with measurements made using DLS technology.

37

(a) Batch EPO-1

(b) Batch S100-1

(c) Batch PLGA-1

Figure 6.5: Representative SEM images of Rhodamine 6G-loaded EPO (a), S100 (b)and PLGA particles (c). There is a wide size distribution for all particle batches. Thesurfaces of the particles in batch S100-1 are not smooth.

38


7.1 Dissolution profiles of Eudragit E PO and PLGAparticlesRhodamine 6G-loaded EPO particles dissolved quickly in pH buffers with pH5.0 and below, as shown in Figure 7.1. For pH 5.0 and below the release ofdye is closely related to the dissolution of the EPO particles. However, in pH7.0 the hydrophilic Rhodamine 6G dye encapsulated inside the Eudragit EPOparticles diffused into the solution prior to dissolution of the EPO particles,generating a positive false initial signal of particle dissolution. In pH 7.0, after6 hours, the EPO particles were still intact, but with no encapsulated dye,see Figure 7.3. After 24 hours, the EPO particles had fully dissolved also inpH 7.0 (data not shown). The calibration curves for the particle dissolutionof Rhodamine 6G-loaded EPO particles, given in Figure 7.4, are overlappingand not pH dependent.

Dissolution profiles of Rhodamine 6G-loaded PLGA particles are given inFigure 7.2. For all pH’s, PLGA particles dissolved at a much slower rate com-pared to EPO particles. The calibration curve for the particle dissolution ofRhodamine 6G-loaded PLGA particles is given in Figure 7.5.

7.2 In vitro uptake of Rhodamine 6G-loaded EudragitE PO particlesThe uptake efficiency of Rhodamine 6G EPO-loaded particles was similar forMSCs and RAW264.7 cells, and not dependent on the initial particle concen-tration (Figure 7.6. Cells incubated for 3 hours with EPO particles took upbetween 9% and 17 % of particles added in the media.

Using fluorescent microscoping technique we investigated the cellular up-take of Rhodamine 6G-loaded EPO particles in MSCs and RAW264.7 cells forup to 6 hours. As illustrated in Figure 7.7 and 7.8, after 2 hours more particlesare associated with cells when the cells were incubated with a higher particleconcentration. Also, it is visible that particles associated with RAW264.7 cellsmost likely dissolute faster and stain the cells by the release of Rhodamine 6Gdye inside the cells.

39

Figure 7.1: Dissolution profiles of Rhodamine 6G-loaded EPO particles in differentpH buffers. The dissolution profile of pH 7.0 is misleading, as the hydrophilic dyeleaked out of the intact EPO particles prior to particle degradation, see Figure 7.3 fordetails. EPO particles dissolved quickly in all buffers with pH 5.0 or below.

7.3 In vitro digestion of Rhodamine 6G-loadedEudragit E PO particlesThe digestion of Rhodamine 6G-loaded EPO particles was lower than the totaluptake of particles in both cell types. After 3 hours, approximately 2-5%, ofthe total amount of particles incubated with cells, had been digested by thecells. MSCs and RAW264.7 cells had similar digestion profiles. Cells digestedEPO particles equally well or better than PLGA particles. See Figure 7.9. Thecalibration curve for the particle dissolution of Rhodamine 6G-loaded EPOand PLGA particles for the in vitro digestion experiment is given in Figure7.10

7.4 Endosomal pH of MSCs and RAW264.7 cellsThe endosomal pH was measured in both LPS activated and non-activatedMSCs and RAW264.7 cells. The pH was higher in endosomes of LPS acti-vated and non-activated RAW264.7 cells, compared to endosomes of MSCs.In both cells types, activating cells with LPS for 4 hours prior to fluorescencemeasurements decreased the endosomal pH by approximately 0.1 pH unit. SeeTable 7.1. The calibration curves used to convert the measured fluorescenceintensity ratios to pH values, are given in Figure 7.11.

Figure 7.12 are representative images, from which the fluorescence intensi-ties were measured. The images are the combined images of the 340 nm and380 nm wavelength channels. All images in Figure 7.12 are of cells incubated

40

Figure 7.2: Dissolution profiles of Rhodamine 6G-loaded PLGA particles in differ-ent pH buffers. In all pH’s, PLGA particles dissolved very slowly compared to Rho-damine 6G-loaded EPO particles.

Table 7.1: Endosomal pH of non-activated and LPS activated MSCs and RAW264.7cells. First value is the average of calculated pH value from 380/340 ratio and340/380 ratio curves (Figure 7.11). Values within parenthesis indicate calculated min-imum and maximum pH values from either of the two ratio curves.

Cell type pH (non-activated) pH (LPS activated)

MSC 4.09 (3.97 - 4.20) 3.96 (3.82 - 4.07)RAW264.7 4.47 (4.43 - 4.51) 4.29 (4.24 - 4.34)

with Rhodamine 6G-loaded EPO particles. LPS-activated RAW264.7 cells arelarger in size than non-activated RAW264.7 cells. MSCs incubated with LPSincreased in size, their cytoskeleton fluoresced with greater intensity and theyhad a higher number of endosomes inside the cells compared to MSCs notincubated with LPS.

7.5 Toxicity of Rhodamine 6G-loaded Eudragit E POparticles in MSCsUsing microscopy, we qualitatively measured the toxicity of Rhodamine 6G-loaded EPO and PLGA particles, by incubation of particles with MSCs cells.Higher concentrations of EPO particles incubated with cells cause higher celldeath rate, as shown in Figure 7.13. Equally high concentrations of PLGA,as EPO particles, particles do not destruct MSCs to the same extent (Figure

41

(a) Particles after 6 hours in pH 4 buffer. Allthe particles have fully dissolved and alldye has been released.

(b) Particles after 6 hours in pH 7 buffer.The dye has diffused out of the particles,whilst most EPO particles are still intact.

Figure 7.3: Rhodamine 6G-loaded EPO particles incubated in pH 4 and 7 buffer so-lutions. After 6 hours, the EPO particles had fully dissolved in low pH buffer, but notin higher pH buffer, though the dye had diffused out of the particles in both solutions.

7.14). Cationic polymers used in gene therapy have previously been shown tobe toxic (Wolfert et al., 1999; Hongtao Lva and Yanc, 2006).

42

Figure 7.4: Calibration curves for dissolution profiles of Rhodamine 6G-loaded EPOparticles. The curves are overlapping and not pH dependent.

Figure 7.5: Calibration curve for dissolution profile of Rhodamine 6G-loaded PLGAparticles. The curve is based on the dissolution of EPO particles in PBS buffer.

43

Figure 7.6: The uptake efficiency of Rhodamine 6G-loaded EPO particles for MSCsand RAW264.7 cells. MSCs and RAW264.7 cells have similar uptake efficiencyacross all measured concentration of EPO particles incubated with cells, ranging from9% to 17% of particles added to media.

44

(a) 0.5 mg EPO particles / ml. Light micro-scope image

(b) 0.5 mg EPO particles / ml. Fluorescentimage.

(c) 0.1 mg EPO particles / ml. Light micro-scope image

(d) 0.1 mg EPO particles / ml. Fluorescentimage.

(e) 0.02 mg EPO particles / ml. Light micro-scope image

(f) 0.02 mg EPO particles / ml. Fluorescentimage.

Figure 7.7: Microscopic images of MSCs incubated with 0.5 (a, b), 0.1 (c, d) and 0.02(e, f) mg EPO particles / ml. Each image is taken after incubating cells for 2 hours at37◦C. There is a higher degree of association between particles and cells with a higherconcentration of particles.

45

(a) 0.5 mg EPO particles / ml. Light micro-scope image

(b) 0.5 mg EPO particles / ml. Fluorescentimage.

(c) 0.1 mg EPO particles / ml. Light micro-scope image

(d) 0.1 mg EPO particles / ml. Fluorescentimage.

(e) 0.02 mg EPO particles / ml. Light micro-scope image

(f) 0.02 mg EPO particles / ml. Fluorescentimage.

Figure 7.8: Microscopic images of RAW264.7 cells incubated with 0.5 (a, b), 0.1 (c,d) and 0.02 (e, f) mg EPO particles / ml. Each image is taken after incubating cellsfor 2 hours at 37◦C. There is a higher degree of association between particles andcells with a higher concentration of particles. Also, most likely, EPO particles havedissolved and released Rhodamine 6G dye inside the cells.

46

Figure 7.9: Digestion efficiency of Rhodamine 6G-loaded EPO and PLGA parti-cles for MSCs and RAW264.7 cells. After 3 hours, approximately 2-5%, of the totalamount of particles incubated with cells, had been digested by the cells. MSCs andRAW264.7 cells had similar digestion profiles. Cells digested EPO particles equallywell or better than PLGA particles.

Figure 7.10: Calibration curve for the dissolution profiles of Rhodamine 6G-loadedEPO and PLGA particles for the in vitro digestion experiment.

(a) 340/380 Ratio as function of pHvalue

(b) 380/340 Ratio as function of pHvalue

Figure 7.11: Calibration curves used to calculate pH values from measured fluores-cence intensities.

47

(a) Non-activated RAW264.7 cells (b) LPS activated RAW264.7 cells

(c) Non-activated MSCs (d) MSCs incubated with LPS

Figure 7.12: RAW264.7 cells and MSCs incubated with and without LPS. These im-ages are representative of those used to determine the ratios between the fluorescenceintensities of wavelengths 340 and 380 nm, using Lysotracker yellow/blue and flu-orescent Rhodamine 6G-loaded particles. The medium sized, strongly fluorescent,circles are Rhodamine 6G-loaded EPO particles. Smaller circles, clearly visible in (c)and (d) are endosomes. When activated with LPS, RAW264.7 cells increase in size,compared to non-activated RAW264.7 cells. MSCs change cell morphology whenincubated with LPS by changing cell shape and size.

48

Figure 7.13: MSCs incubated with different concentrations of Rhodamine 6G-loadedEPO particles at different time points. After 4, 5 and 6 hours, cells incubated withhigher concentrations of Rhodamine 6G-loaded particles show a higher rate of celldeath; indicated by the destruction of the cells.

(a) 0.1 mg PLGA / ml. Light micro-scope image.

(b) 0.1 mg PLGA / ml. Fluorescentimage.

(c) 0.5 mg PLGA / ml. Light micro-scope image.

(d) 0.5 mg PLGA / ml. Fluorescentimage.

Figure 7.14: MSCs incubated with different concentrations of Rhodamine 6G-loadedPLGA particles at 27 hours. It is clear that the PLGA particles have not adverselyaffected the MSCs at concentrations as high as 0.5 mg particles / ml.

49


8.1 Characterization of iron oxide-loaded particles8.1.1 Size distributions of Rhodamine 6G-loaded particlesThe average diameter of the EPO-I and EPO-D particles batches are given inTable 8.1.

8.1.2 TEM images of iron oxide-loaded Eudragit E PO particlesTEM images of iron oxide- and iron oxide- & DiO-loaded Eudragit EPO par-ticles are given in Figure 8.1. iron oxide-loaded EPO particles with a higherinitial amount of iron oxide in the synthesis process, yielded a higher concen-tration of iron oxide particles inside the EPO nanoparticles.

8.1.3 Zeta potential of iron oxide-loaded EPO and PLGAparticlesAs indicated by Figure 8.2, the zeta potential is positive for iron oxide-loadedEPO particles for pH 5 to 8. The EPO particles dissolved in pH below 5. Ironoxide-loaded PLGA particles have a negative zeta potential for pH above 4.

Table 8.1: Synthesized iron oxide- and iron oxide &DiO-loaded particles and theirrespective size distributions, given by DLS.

Batch ID Diameter (nm)

EPO-I 335 ± 9EPO-D 1132 ± 267

50

8.2 In vitro uptake of iron oxide- & DiO-loadedEudragit E PO particlesUsing FACS technology, we measured the uptake of iron oxide- & DiO-loadedEudragit E PO particles in MSCs. Cells incubated with EPO particles hadan increased DiO signal, indicating these cells take up the particles (Figure8.3(a)). There was no increase in uptake when loading EPO particles withpositively charged PLL particles. Most likely PLL did not bind to the surfaceof the EPO particles. Also, by comparing with the intensity signal of DiO dyeinside EPO particles (Figure 8.3(b)), we can conclude that cells, on average,take up more than one particle.

8.3 Confocal microscopy of cells incubated with ironoxide- & DiO-loaded Eudragit E PO particlesShown in Figure 8.4, are images taken using confocal microscopic technique.Membrane structures inside MSCs were labelled with DiO, indicating thatDiO dye leaked out from the particles. This does not indicate whether ironoxide particles were released or not. The cell membrane staining was not suc-cessful as there is very low red signal. RAW264.7 cell membranes were moresuccessfully stained. Although the cell membrane staining was not uniform,it is clear that iron oxide- & DiO-loaded EPO particles (green dots) are in-side the RAW264.7 cells. Similar to MSCs, the membranes inside the cellcytoplasms were stained with DiO, suggesting that iron oxide- & DiO-loadedEPO particles entered the cells and that dye leaked out from the particles.

8.4 Toxicity of iron oxide- & DiO-loaded Eudragit EPO particles in MSCs and RAW264.7 cellsUsing a quantitative method, after 3 hours, the cell viability was higher than70% when incubating MSCs and RAW264.7 cells with iron oxide- & DiO-loaded EPO particles with concentrations up to 0.05 mg/ml (Figure 8.5).

51

(a) Iron oxide-loaded EPO particles. These particleswere synthesised using 24.9 mg iron oxide.

(b) Iron oxide- & DiO-loaded EPO particles. The parti-cles were synthesised using 4.8 mg iron oxide.

Figure 8.1: TEM images of iron oxide- and iron oxide- & DiO-loaded EPO particles.Particles made with higher iron oxide (a) had a higher content of iron oxide comparedto particles made with a lower initial amount of iron oxide (b).

52

Figure 8.2: Zeta potential of iron oxide-loaded EPO and PLGA particles. The EPOparticles are positively charged for pH 5 to 8 and PLGA particles are positivelycharged for pH below 4 and negatively charged for pH above 4.

(a) DiO signal from MSCs incubated withiron oxide- & DiO-loaded Eudragit E POparticles

(b) DiO signal from iron oxide- & DiO-loaded Eudragit E PO particles

Figure 8.3: FACS of MSCs incubated without particles, with EPO particles and alsowith EPO-PLL particles. In (a), Blue indicates MSCs labelled with iron oxide- &DiO-loaded Eudragit E PO particles, red indicates MSCs labelled with EPO-PLL par-ticles, and black indicates unlabelled MSCs. This data clearly indicates a higher DiOsignal in cells incubated with EPO particles. Also the signal is similar for cells incu-bated with EPO and EPO-PLL particles. In (b), the DiO signal intensity for individualEPO particles is lower than that of individual cells in (a), indicating that each cell, onaverage, take up more than one EPO particle.

53

(a) MSC

(b) RAW264.7 cells

Figure 8.4: Confocal microscopic images of MSCs and RAW264.7 cells incubatedwith iron oxide- & DiO-loaded EPO particles. In (a) and (b), membrane structuresinside MSCs and RAW264.7 cells were labelled with DiO, indicating DiO dye leakedout from the particles. In (b), it is clear that iron oxide- & DiO-loaded EPO particles(green dots) are inside the RAW264.7 cells, suggesting that iron oxide- & DiO-loadedEPO particles entered the cells. The red color is DiD staining of the cell membranes.

54

Figure 8.5: Cell viability of MSCs and RAW264.7 cells, after incubating cells withiron oxide- & DiO-loaded EPO particles for 3 hours. The cell viability was higherthan 70% when incubating MSCs and RAW264.7 cells with iron oxide- & DiO-loadedEPO particles with concentrations up to 0.05 mg/ml.

55

Part IV:

Discussion


9.1 Preparation of Rhodamine 6G-loaded particlesThere are numerous methods for preparing and synthesizing nano- and mi-croparticles (Pinto Reis et al., 2006). The protocols we employed in this studyhad previously been proven to synthesize most of the Eudragit polymers usedin this study. We extended previous studies by successfully encapsulatingRhodamine 6G into Eudragit E PO, S 100, L 100-55 and L 100 polymers.

9.2 Characterization of Rhodamine 6G-loadedparticlesThe synthesized particles differed in size, depending on the protocol used.The effect of the IOP phase, either D-E-I mix or MeOH, was not significantat a concentration of 10% PVA in the IAP, when using 3% polymer in theIOP. However, when using MeOH as IOP and 0.5% polymer, the size of theparticles decreased significantly. Previous studies have shown, that an optimalaverage diameter for transfection is around 100 nm (Mintzer and Simanek,2009; Aoyama et al., 2003; Gao et al., 2005). We conclude that using MeOHas IOP is more suitable for gene delivery vessel design, as this decreases theparticle size. Most commonly, previous studies have used dispersing agent (inthis study PVA) concentrations between 1-3% in the IAP (Jung et al., 2007;Esposito et al., 1999; Jain et al., 2005). We observed a local minimum ofparticles sizes using 10% of PVA. This phenomenon has been previously doc-umented (Fu et al., 2003). A smaller amount of polymer in the IOP decreasedthe particle size, this is also supported in work done by C. Bothiraja and Sher(2009). It was not surprising that longer centrifugation time decreased averageparticle diameter, as an increase in time will allow a larger number of smallerparticles to reach the pellet while centrifuging, thus decreasing the averagemeasured diameter. Variations in measured average particle diameter betweenDLS and Coulter Counter can be explained by the fact that these instrumentshave different optimal ranges, within which they can measure particle sizes.Particles of sizes in the optimal measurable range for both machines had sim-ilar measured average diameter. The Coulter Counter provides graphical andnumerical output. The mean numeric output does not always necessarily cor-

57

respond with the graphical top peak. Sometimes the median would have beena better estimate of the actual top peak, however we decided to provide thenumerical mean, though we are aware this does somewhat skew the measuredsize, this because the Coulter Counter could not measure the smaller particles.

SEM images of EPO, S100 and PLGA particles show a wide distributionin particle size. This wide range in particle size was unexpected, and has not,to our knowledge, been reported elsewhere. This, however, does lead us to theconclusion that the particle size can be easily fine tuned by centrifuging first atdesired low speed to remove large particles and then at higher speed to capturesmaller particles. Of course, this does decrease the yield of particles from eachsynthesized batch. On average, 10% of the amount of synthesized particleswere in the described size ranges and used in this study (data not shown). Thewide size distribution of particles, shown in the SEM images, correlates wellwith wide distributions of average diameters of particles, measured by DLS.

58


10.1 Dissolution profiles of Eudragit E PO and PLGAparticlesThe dissolution profiles of EPO particles correlates well with specificationsof the Eudragit E PO polymer by the manufacturer (Evonik-Industries, 2010).E PO polymer is swellable in pH above 5. The release of Rhodamine 6Gdye from dissolved particles is pH dependent an faster at lower pH, with adistinct difference in release between most pH’s (except 4.5 and 5) for the firsthour (Figure 7.1). Dissolutions experiments with L100-55 showed similar, butreversed, pH dependence. The L100-55 dissolution profiles also correlatedwell with the manufacturers description of the polymer’s pH dependency (datanot shown). However, it was not expected to observe any dissolution of EPOparticles in pH 7.0. First, hydrophilic dye leaked out of the particles givingrise to a misleading profile of particle dissolution. The dissolution of EPOparticles in pH 7.0 is, however, much slower compared to the dissolution inbuffers with lower pH.

The pH independence of the EPO calibration curve for particle dissolutionmade it possible to use one combined curve for the conversion of intensityinto mg.

As control, we also tested the dissolution of PLGA particles. The PLGAparticles dissolved at a much slower rate, compared to the EPO particles. Theslow dissolution of PLGA particles is well known (Liu et al., 2010). This sup-ports that our system gives valid measurements of particle dissolution whenthe particles dissolve without prior release of hydrophilic dye.

The calibration curve for the dissolution of PLGA particles was based onmeasurements made with EPO particles. The calibration curve and dissolutionprofiles were based on single measurements. Reasons for this design, was onlytime constraints. However, in the next step of the project we will redo thesemeasurements in triplicate and base the calibration curve on the dissolution ofPLGA rather than EPO particles. However, we do not consider this to affectthe overall conclusion of the slow PLGA particle dissolution.

59

10.2 In vitro uptake and digestion of Rhodamine6G-loaded Eudragit E PO particlesThe uptake and digestion of Rhodamine 6G-loaded EPO particles was inves-tigated in MSCs and RAW264.7 cells, both qualitatively and quantitatively.The uptake efficiency of EPO particles, 9-17% was similar in both cell linesand somewhat lower than presented by other studies for PLGA particles (20-30% uptake efficiency) (Liu et al., 2010). There was no significant difference,comparing different incubation concentration of particles. The digestion ofparticles was, as expected, lower than the total uptake (2-5%). This indicatethat, after 3 hours, only a small amount of particles have been internalizedby the cells. There was no significant difference in particle digestion betweenthe cell lines. This was unexpected, as we believed RAW264.7 cells would di-gest particles quicker. However, a quicker digestion of particles in RAW264.7cells was supported by the microscopic images, clearly showing a higher dis-solution of particles in RAW264.7 cells compared to MSCs, by observing thatentire cells became fluorescent.

The protocols for measuring the uptake and digestion of particles was basedon previous work by Harfouche et al. (2009). However, both these protocolssuffer several drawbacks. Also, the authors of (Harfouche et al., 2009) agreethat the used protocol is not optimal (personal communication). The mainproblem is that it cannot be determined whether the particles are inside thecells, or attached on the cell surface, thus the uptake experiment, rather in-dicates particles associated with the cells, and not particles taken up by thecells. In the digestion experiment, the actual particle digestion is most likelyhigher than measured because, throughout the experiment, not all fluorescentmolecules are collected in the centrifugation step. Also, there is no measurefor how efficient the cell lysis method is. If not all cells are lysed, only afraction of dye from the digested particles is released into the supernatant.Furthermore, heating does affect the fluorescence intensity of Rhodamine 6Gby fading the fluorescence of the dye (H.T. Oh and Yun, 1992).

The digestion of PLGA particles in cells is similar to that of EPO particles,indicating that the uptake of PLGA particles is comparable to that of EPOparticles. We find it intriguing that PLGA particles are digested equal to EPOparticles. This especially as the dissolution profile of PLGA particles indicatesthat PLGA particles do not dissolve quickly in pH buffer with equivalent pH ofthat of cellular endosomes or cytosol. Also, previous studies have shown thatPLGA particles are not internalized equally well (Basarkar and Singh, 2009).We conclude that, this result is most likely an artefact of the experiment setup.

The amount of dissolved PLGA particles, in the dissolution experiment,was calculated using a calibration curve based on dissolution of EPO particles.This because of time constraints. However, in future work, the calculationswill be based on a calibration curve for PLGA particles. However, we do notexpect this to affect the overall conclusion that these experiment indicate that

60

EPO and PLGA particles are taken up and digested equally, though we do notfully discard this possibility.

10.3 Endosomal pH of MSCs and RAW264.7 cellsThe endosomal pH of MSCs and RAW264.7 cells, incubated with and with-out LPS, was measured using standard ratiometric imaging technique. Theendosomal pH of MSCs was lower compared to that of RAW264.7 cells incells incubated both with and without LPS. The calculated pH inside the en-dosomes of the cell types ranged from a minimum 3.82 to a maximum 4.51.These measurements are significantly lower than what previous studies haveshown (5.0-6.4) (Ohkuma and Poole, 1978; Jiang et al., 1990). Explanationsfor this estimated low pH, could be because of difficulties correctly selectingendosomes displayed on the computer screen or incorrect pH dependency ofthe fluorescent dye at the measured wavelengths. It is important to measurethe fluorescence intensity at one wavelength, which is strongly affected bypH, and one wavelength not affected by pH (Holopainen et al., 2001). How-ever, at a pH below 4.5 and in the pH range around 5.0, EPO particles dissolvequickly. A low endosomal pH will allow EPO particles to quickly dissolve andrelease associated molecules (e.g. DNA). If the endosomal pH is around 6.4,EPO particles will still dissolve, in accordance with previous results, but at aslower rate. Our results indicate that the endosomal pH is cell type dependent.

10.4 Toxicity of Rhodamine 6G-loaded Eudragit E POparticles in MSCsMicroscopic images of MSCs incubated with different concentrations of EPOparticles, taken at half an hour intervals, indicate that EPO particles are toxicto cells. The toxicity of EPO particles is especially notable at concentrationshigher than 20 µg/ml. Most likely, higher particle toxicity at higher concentra-tions, is linked to a higher uptake of particles per cell. Measuring the toxicityby microscopy is not a quantitative method, though it does provide some in-sight to how toxic the particles are. Comparing results from incubating cellswith EPO and PLGA particles, it is evident that EPO particles are more toxicthan PLGA particles.

61


11.1 Characterization of iron oxide-loaded particlesIron oxide-loaded EPO particles (EPO-I) had a low variation in size with anaverage diameter of approximate 335 nm, this is in accordance with previouslypublished results (Liu et al., 2007a). The EPO-D particles were measured to bemuch larger. However, we do not believe these measurements to be accuratebecause these particles most likely did not fully dissolve into the water, thushaving aggregated and formed larger particles. Similar measurements madewith EPO-I showed the same pattern with aggregation of particles. TEM im-ages confirm the size of the EPO-I particles. It was possible to control theloading of iron oxide inside particles depending on the amount of iron oxideused in the synthesis steps. The particles were uniform in shape.

The zeta potential of iron oxide-loaded EPO particles was highly positivein all measured pH buffers, indicating that the EPO particles are positivelycharged. This is a strong feature of this system. Positively charged particleseasily interact with cell surfaces and are more easily taken up by cells, com-pared to negatively charged particles (Basarkar and Singh, 2009). However,a positive surface charge on compounds also increases their toxicity. PLGAparticles, however, are negatively charged in pH buffers above 4. These find-ings support the decreased toxicity and decreased uptake of PLGA particlescompared to EPO particles (indicated by microscopic images in Figure 7.13and 7.14). Also, a positively charged surface of the EPO particles support theobserved increased toxicity of particles. The measured zeta potential of PLGAis comparable to that measured by Panyam et al. (2002), indicating that ourzeta potential measurements are valid.

11.2 In vitro uptake of iron oxide- & DiO-loadedEudragit E PO particlesResults from FACS experiments show that MSCs incubated with iron oxide-& DiO-loaded EPO particles take up particles. Also, by comparing the flu-orescence intensities of measured cells and pure particles, it is evident thatcells, on average, take up more than one particle per cell. EPO particles wereincubated with positively charged PLL to form EPO-PLL complexes. These

62

complexes were incubated together with cells. There was no significant in-crease in cellular uptake of EPO-PLL complexes, compared to EPO particles.This indicates that EPO particles are positively charged and increasing theirpositive charge further, does not increase the cellular uptake. Using FACS toinvestigate cellular uptake of particles, is a common method, and have pre-viously been employed by Kim et al. (2010). Still, this method also, as ourpreviously used method, cannot distinguish between particles inside the cells,or attached to the cell surface.

11.3 Confocal microscopy of cells incubated with ironoxide- & DiO-loaded Eudragit E PO particlesConfocal microscopic images of MSCs and RAW264.7 cells incubated withEPO particles, show that RAW264.7 cells take up EPO particles and that DiOdye leaks out from particles, most likely due to particle dissolution, stainingboth MSCs and RAW264.7 cells. These findings support our previous exper-iments, that cells do take up and digest EPO particles. The DiD staining inthe confocal microscopic experiment was not very successful, however, wedo not believe this affects the overall conclusion that cells take up and digestparticles. Confocal microscopy has previously been used to show location ofparticles inside cells by Jung et al. (2007).

11.4 Toxicity of iron oxide- & DiO-loaded Eudragit EPO particles in MSCs and RAW264.7 cellsUsing an MTT assay, the toxicity of iron oxide- & DiO-loaded EPO parti-cles was measured in MSCs and RAW264.7 cells. The cell viability was morethan 70% for both cells types after incubating cells for 3 hours. The maximumtested concentration, 50 µg, is used in other studies, and cells incubated withthis amount of PLGA nanoparticles have a viability higher than 55% after 48hours (Prashant et al., 2010). 3 hours is a very short time for measuring cellviability, and in future studies we aim to increase the incubation time. Thesefindings support our previous observations that high concentrations of EPOparticles increase cell death, however, Flora et al. (2008) and C. Bothiraja andSher (2009) have previously shown that Eudragit E PO polymer and nanopar-ticles made with Eudragit E PO polymer have low toxicity.

63

Part V:

Conclusions and Future Work

12. Conclusions and Future Work

We have successfully synthesized nanoparticles using four differentEudragit polymers and also using PLGA polymer, by employing noveldouble-emulsion techniques. We have been able to control the size andmorphology of these synthesized particles by selecting centrifugation time,concentration of surfactant in the aqueous phase, type of organic phase andby changing initial polymer concentration. The dissolution of synthesizedEudragit polymer nanoparticles can be controlled, and we have verified slowdissolution of PLGA particles in all measured pH buffers. Cellular uptakeand digestion has been investigated, and it has been shown that EPO particlesare taken up and digested equally well, or more efficiently, compared toPLGA particles. The endosomal pH of MSCs and RAW264.7 cells has beenmeasured and the toxicity of EPO particles investigated.

Furthermore, we have synthesized iron oxide-loaded and iron oxide- &DiO-loaded nanoparticles using a sonication-emulsification method. We haveshown that it is possible to control the amount of encapsulated iron oxide par-ticles inside the EPO particles. We have verified previous measurements of thesurface charge of PLGA particles in different pH buffers, and also observedthat EPO particles are positively charged in any pH. It has been shown thatMSCs, on average, take up more than one EPO particle per cell, and also thatRAW264.7 cells take up EPO particles. Finally we have shown no significantdifference in toxicity of iron oxide & DiO-loaded EPO particles in MSCs andRAW264.7 cell lines.

It can be concluded that positively charged, nanosized and ironoxide-loaded Eudragit E PO particles are stable in high pH solutions and willquickly dissolve once inside the endosomes of cells. These nanoparticleshave great potential as carriers to transport DNA plasmids into the nucleus ofcells using magnetic homing technique.

Future work includes extending the system of pure EPO particles to EPO-PLGA complex particles. This will most likely decrease the toxicity of the par-ticles. Also, we intend to synthesize EPO-DNA and EPO-PLGA-DNA com-plexes to study the transfection of DNA using these two types of carriers. Afinal step is to, in vitro, investigate the homing capabilities of EPO-DNA andEPO-PLGA-DNA complexes with encapsulated iron oxide, to investigate theincreased efficacy of a magnetic homing system, compared to a system with-out homing abilities.

65

Part VI:

Acknowledgements

Acknowledgements

I wish to thank Jeffrey Karp for giving me this opportunity to pursue my lifelong dream, to experience and be involved in cutting edge science at one ofthe world’s most prestigious institutes. I am very grateful to Chenjie Xu, forthe guidance and help he has offered me throughout this project. Chenjie hashelped me increase my scientific knowledge and shown me new ways to thinkabout science.

I wish to thank my closest collaborators Mary Mu, for introducing me tothe project and helping my first months in the lab, and David Miranda-Nievesfor continuing on where I left off.

A special thank you to Weian Zhao, for helping with ideas on gene transfec-tion, and to Sudipta Basu, for assisting in the borrowing of equipment fromSengupta’s laboratory. I also wish to thank all the other collaborators fromother labs for assisting me in the use of their equipment and for providinghelpful feedback. A special thank you to Amy Blass from Soybel’s laboratoryfor her kind help with the endosomal pH measurements.

I wish to thank Jöns Hilborn for accepting the position as my scientificreviewer. and helping me when I needed it. And last, but not least, I wish tothank Margareta Krabbe for her unforgettable help in assisting me, in all of myscientific endeavours. These have, to date, taken me to participate in researchprojects in almost all continents of the world.

67

Part VII:

Bibliography

Bibliography

Morten Ø Andersen, Agata Lichawska, Ayyoob Arpanaei, Stig MøllerRask Jensen, Harpreet Kaur, David Oupicky, Flemming Besenbacher, PeterKingshott, Jørgen Kjems, and Kenneth A Howard. Surface functionalisationof plga nanoparticles for gene silencing. Biomaterials, 31(21):5671–7, Jul2010. doi: 10.1016/j.biomaterials.2010.03.069.

D Ang, Q V Nguyen, S Kayal, P R Preiser, R S Rawat, and R V Ramanujan.Insights into the mechanism of magnetic particle assisted gene delivery. ActaBiomater, Oct 2010. doi: 10.1016/j.actbio.2010.09.037.

Yasuhiro Aoyama, Takuya Kanamori, Takashi Nakai, Toshinori Sasaki,Shohei Horiuchi, Shinsuke Sando, and Takuro Niidome. Artificial virusesand their application to gene delivery. size-controlled gene coating with gly-cocluster nanoparticles. J Am Chem Soc, 125(12):3455–7, Mar 2003. doi:10.1021/ja029608t.

Ashwin Basarkar and Jagdish Singh. Poly (lactide-co-glycolide)-polymethacrylate nanoparticles for intramuscular delivery of plasmid encod-ing interleukin-10 to prevent autoimmune diabetes in mice. Pharm Res, 26(1):72–81, Jan 2009. doi: 10.1007/s11095-008-9710-4.

Jean Paul Behr. Synthetic gene-transfer vectors. Accounts of Chemical Re-search, 26(5):274–8, 1993.

Jamie M Bergen, In-Kyu Park, Philip J Horner, and Suzie H Pun. Nonviralapproaches for neuronal delivery of nucleic acids. Pharm Res, 25(5):983–98,May 2008. doi: 10.1007/s11095-007-9439-5.

Dhruba J Bharali, Ilona Klejbor, Ewa K Stachowiak, Purnendu Dutta, IndrajitRoy, Navjot Kaur, Earl J Bergey, Paras N Prasad, and Michal K Stachowiak.Organically modified silica nanoparticles: a nonviral vector for in vivo genedelivery and expression in the brain. Proc Natl Acad Sci U S A, 102(32):11539–44, Aug 2005. doi: 10.1073/pnas.0504926102.

Ugo Bilati, Eric Allémann, and Eric Doelker. Sonication parameters forthe preparation of biodegradable nanocapsules of controlled size by thedouble emulsion method. Pharm Dev Technol, 8(1):1–9, 2003. doi:10.1081/PDT-120017517.

69

Savita Bisht, Georg Feldmann, Sheetal Soni, Rajani Ravi, Collins Karikar,Amarnath Maitra, and Anirban Maitra. Polymeric nanoparticle-encapsulatedcurcumin ("nanocurcumin"): a novel strategy for human cancer therapy. JNanobiotechnology, 5:3, 2007. doi: 10.1186/1477-3155-5-3.

Bas Blits, Sanne Derks, Jaap Twisk, Erich Ehlert, Jolanda Prins, and JoostVerhaagen. Adeno-associated viral vector (aav)-mediated gene transfer in thered nucleus of the adult rat brain: comparative analysis of the transductionproperties of seven aav serotypes and lentiviral vectors. J Neurosci Methods,185(2):257–63, Jan 2010. doi: 10.1016/j.jneumeth.2009.10.009.

W M Bonner. Protein migration into nuclei. i. frog oocyte nuclei in vivo ac-cumulate microinjected histones, allow entry to small proteins, and excludelarge proteins. J Cell Biol, 64(2):421–30, Feb 1975.

M Briones, M Singh, M Ugozzoli, J Kazzaz, S Klakamp, G Ott, andD O’Hagan. The preparation, characterization, and evaluation of cationic mi-croparticles for dna vaccine delivery. Pharm Res, 18(5):709–12, May 2001.

Nicola Brunetti-Pierri and Philip Ng. Progress towards liver and lung-directed gene therapy with helper-dependent adenoviral vectors. Curr GeneTher, 9(5):329–40, Oct 2009.

Karimunnisa S. Shaikh C. Bothiraja, Atmaram P. Pawar and Praveen Sher.Eudragit epo based nanoparticle suspension of andrographolide: in vitro andin vivo. Nanosci. Nanotechnol. Lett., 1:156–164, 2009.

Walter R. Carmody. Easily prepared wide range buffer series. J. Chem.Educ., 38(11):559–560, 1961.

M Cavazzana-Calvo, S Hacein-Bey, G de Saint Basile, F Gross, E Yvon,P Nusbaum, F Selz, C Hue, S Certain, J L Casanova, P Bousso, F L Deist,and A Fischer. Gene therapy of human severe combined immunodeficiency(scid)-x1 disease. Science, 288(5466):669–72, Apr 2000.

Patrice Chollet, Marie C Favrot, A Hurbin, and Jean-Luc Coll. Side-effectsof a systemic injection of linear polyethylenimine-dna complexes. J GeneMed, 4(1):84–91, 2002.

Rita Cortesi, Carlo Mischiati, Monica Borgatti, Laura Breda, AlessandraRomanelli, Michele Saviano, Carlo Pedone, Roberto Gambari, and Clau-dio Nastruzzi. Formulations for natural and peptide nucleic acids based oncationic polymeric submicron particles. AAPS PharmSci, 6(1):E2, Jan 2004.doi: 10.1208/pt060102.

70

Jundong Dai, Tsuneji Nagai, Xueqing Wang, Tao Zhang, Meng Meng, andQiang Zhang. ph-sensitive nanoparticles for improving the oral bioavail-ability of cyclosporine a. Int J Pharm, 280(1-2):229–40, Aug 2004. doi:10.1016/j.ijpharm.2004.05.006.

A Darji, C A Guzmán, B Gerstel, P Wachholz, K N Timmis, J Wehland,T Chakraborty, and S Weiss. Oral somatic transgene vaccination using at-tenuated s. typhimurium. Cell, 91(6):765–75, Dec 1997.

C de Duve, T de Barsy, B Poole, A Trouet, P Tulkens, and F Van Hoof.Commentary. lysosomotropic agents. Biochem Pharmacol, 23(18):2495–531, Sep 1974.

M P Desai, V Labhasetwar, E Walter, R J Levy, and G L Amidon. Themechanism of uptake of biodegradable microparticles in caco-2 cells is sizedependent. Pharm Res, 14(11):1568–73, Nov 1997.

Padma V Devarajan and Ganeshchandra S Sonavane. Preparation and invitro/in vivo evaluation of gliclazide loaded eudragit nanoparticles as a sus-tained release carriers. Drug Dev Ind Pharm, 33(2):101–11, Feb 2007. doi:10.1080/03639040601096695.

Z Diwu, C S Chen, C Zhang, D H Klaubert, and R P Haugland. A novelacidotropic ph indicator and its potential application in labeling acidic or-ganelles of live cells. Chem Biol, 6(7):411–8, Jul 1999.

Nicolas Duceppe and Maryam Tabrizian. Advances in using chitosan-based nanoparticles for in vitro and in vivo drug and gene delivery. ExpertOpin Drug Deliv, 7(10):1191–207, Oct 2010. doi: 10.1517/17425247.2010.514604.

E Esposito, S Sebben, R Cortesi, E Menegatti, and C Nastruzzi. Preparationand characterization of cationic microspheres for gene delivery. Int J Pharm,189(1):29–41, Oct 1999.

Evonik-Industries. Eudragit e po, October 2010. URLhttp://eudragit.evonik.com/product/eudragit/en/products-services/eudragit-products/protective-formulations/e-po/Pages/default.aspx.

FDA. Inactive ingredient search, October 2010. URL http://www.accessdata.fda.gov.

C Featherstone, M K Darby, and L Gerace. A monoclonal antibody againstthe nuclear pore complex inhibits nucleocytoplasmic transport of protein andrna in vivo. J Cell Biol, 107(4):1289–97, Oct 1988.

71

P L Felgner, T R Gadek, M Holm, R Roman, H W Chan, M Wenz, J PNorthrop, G M Ringold, and M Danielsen. Lipofection: a highly efficient,lipid-mediated dna-transfection procedure. Proc Natl Acad Sci U S A, 84(21):7413–7, Nov 1987.

Joseph R V Flora, Benjamin Baker, Daniel Wybenga, Huiying Zhu, andC Marjorie Aelion. Preparation of acidic and alkaline macrocapsulesfor ph control. Chemosphere, 70(6):1077–84, Jan 2008. doi: 10.1016/j.chemosphere.2007.07.060.

T Friedmann. Human gene therapy–an immature genie, but certainly out ofthe bottle. Nat Med, 2(2):144–7, Feb 1996.

Karen Fu, Roy Harrell, Kim Zinski, Christina Um, Ana Jaklenec, Jan Fra-zier, Noah Lotan, Paul Burke, Alexander M Klibanov, and Robert Langer. Apotential approach for decreasing the burst effect of protein from plga micro-spheres. J Pharm Sci, 92(8):1582–91, Aug 2003. doi: 10.1002/jps.10414.

Feilim M Gabhann, Brian H Annex, and Aleksander S Popel. Gene therapyfrom the perspective of systems biology. Curr Opin Mol Ther, 12(5):570–7,Oct 2010.

Huajian Gao, Wendong Shi, and Lambert B Freund. Mechanics of receptor-mediated endocytosis. Proc Natl Acad Sci U S A, 102(27):9469–74, Jul 2005.doi: 10.1073/pnas.0503879102.

M Gargouri, A Sapin, S Bouli, P Becuwe, J L Merlin, and P Maincent. Op-timization of a new non-viral vector for transfection: Eudragit nanoparticlesfor the delivery of a dna plasmid. Technol Cancer Res Treat, 8(6):433–44,Dec 2009.

Ulrich Reinhart Goessler, Katrin Riedel, Karl Hormann, and Frank Riedel.Perspectives of gene therapy in stem cell tissue engineering. Cells TissuesOrgans, 183(4):169–79, 2006. doi: 10.1159/000096508.

Christine Gonçalves, Eric Mennesson, Renate Fuchs, Jean-Pierre Gorvel,Patrick Midoux, and Chantal Pichon. Macropinocytosis of polyplexes andrecycling of plasmid via the clathrin-dependent pathway impair the transfec-tion efficiency of human hepatocarcinoma cells. Mol Ther, 10(2):373–85,Aug 2004. doi: 10.1016/j.ymthe.2004.05.023.

Rania Harfouche, Sudipta Basu, Shivani Soni, Dirk M Hentschel, Raghu-nath A Mashelkar, and Shiladitya Sengupta. Nanoparticle-mediated targetingof phosphatidylinositol-3-kinase signaling inhibits angiogenesis. Angiogen-esis, 12(4):325–38, 2009. doi: 10.1007/s10456-009-9154-4.

72

Tanya S Hauck, Arezou A Ghazani, and Warren C W Chan. Assess-ing the effect of surface chemistry on gold nanorod uptake, toxicity, andgene expression in mammalian cells. Small, 4(1):153–9, Jan 2008. doi:10.1002/smll.200700217.

M A Hickman, R W Malone, K Lehmann-Bruinsma, T R Sih, D Knoell, F CSzoka, R Walzem, D M Carlson, and J S Powell. Gene expression followingdirect injection of dna into liver. Hum Gene Ther, 5(12):1477–83, Dec 1994.doi: 10.1089/hum.1994.5.12-1477.

J M Holopainen, J Saarikoski, P K Kinnunen, and I Järvelä. Elevated lyso-somal ph in neuronal ceroid lipofuscinoses (ncls). Eur J Biochem, 268(22):5851–6, Nov 2001.

Bing Wang Shaohui Cui Hongtao Lva, Shubiao Zhang and Jie Yanc. Tox-icity of cationic lipids and cationic polymers in gene delivey. Journal ofControlled Release, 114(1):100–109, August 2006.

T.Y. Kwona B.K. Moon H.T. Oh, Hyang-Soon Kam and S.I. Yun. Fluores-cence behavior of rhodamine 6g doped sol-gel glasses at low temperature.Materials Letters, 13(2-3):139–141, March 1992.

Hongliang Huang, Hai Yu, Guping Tang, Qingqing Wang, and Jun Li.Low molecular weight polyethylenimine cross-linked by 2-hydroxypropyl-gamma-cyclodextrin coupled to peptide targeting her2 as a gene deliveryvector. Biomaterials, 31(7):1830–8, Mar 2010. doi: 10.1016/j.biomaterials.2009.11.012.

Deepti Jain, Amulya K Panda, and Dipak K Majumdar. Eudragit s100 en-trapped insulin microspheres for oral delivery. AAPS PharmSciTech, 6(1):E100–7, 2005. doi: 10.1208/pt060116.

L W Jiang, V M Maher, J J McCormick, and M Schindler. Alkalinizationof the lysosomes is correlated with ras transformation of murine and humanfibroblasts. J Biol Chem, 265(9):4775–7, Mar 1990.

Jua Jung, In-Hyun Lee, Eunhye Lee, Jinho Park, and Sangyong Jon. ph-sensitive polymer nanospheres for use as a potential drug delivery vehicle.Biomacromolecules, 8(11):3401–7, Nov 2007. doi: 10.1021/bm700517z.

Nadine Wong Shi Kam, Zhuang Liu, and Hongjie Dai. Functionalizationof carbon nanotubes via cleavable disulfide bonds for efficient intracellulardelivery of sirna and potent gene silencing. J Am Chem Soc, 127(36):12492–3, Sep 2005. doi: 10.1021/ja053962k.

Michael G Kaplitt, Andrew Feigin, Chengke Tang, Helen L Fitzsimons, PaulMattis, Patricia A Lawlor, Ross J Bland, Deborah Young, Kristin Strybing,

73

David Eidelberg, and Matthew J During. Safety and tolerability of genetherapy with an adeno-associated virus (aav) borne gad gene for parkinson’sdisease: an open label, phase i trial. Lancet, 369(9579):2097–105, Jun 2007.doi: 10.1016/S0140-6736(07)60982-9.

Forrest M Kievit, Omid Veiseh, Chen Fang, Narayan Bhattarai, DonghoonLee, Richard G Ellenbogen, and Miqin Zhang. Chlorotoxin labeled magneticnanovectors for targeted gene delivery to glioma. ACS Nano, 4(8):4587–94,Aug 2010. doi: 10.1021/nn1008512.

Jae-Hwan Kim, Ji Sun Park, Han Na Yang, Dae Gyun Woo, Su Yeon Jeon,Hyun-Jin Do, Hye-Young Lim, Jung Mo Kim, and Keun-Hong Park. The useof biodegradable plga nanoparticles to mediate sox9 gene delivery in humanmesenchymal stem cells (hmscs) and induce chondrogenesis. Biomaterials,Sep 2010. doi: 10.1016/j.biomaterials.2010.08.086.

R M Klein, E D Wolf, R Wu, and J C Sanford. High-velocity microprojectilesfor delivering nucleic acids into living cells. 1987. Biotechnology, 24:384–6,1992.

Cédric Klumpp, Kostas Kostarelos, Maurizio Prato, and Alberto Bianco.Functionalized carbon nanotubes as emerging nanovectors for the deliveryof therapeutics. Biochim Biophys Acta, 1758(3):404–12, Mar 2006. doi:10.1016/j.bbamem.2005.10.008.

C Kneuer, M Sameti, E G Haltner, T Schiestel, H Schirra, H Schmidt, andC M Lehr. Silica nanoparticles modified with aminosilanes as carriers forplasmid dna. Int J Pharm, 196(2):257–61, Mar 2000.

Ling Kong and Bao-Xue Ge. Myd88-independent activation of a novel actin-cdc42/rac pathway is required for toll-like receptor-stimulated phagocytosis.Cell Res, 18(7):745–55, Jul 2008. doi: 10.1038/cr.2008.65.

Idit Kopatz, Jean-Serge Remy, and Jean-Paul Behr. A model for non-viralgene delivery: through syndecan adhesion molecules and powered by actin.J Gene Med, 6(7):769–76, Jul 2004. doi: 10.1002/jgm.558.

Rasti Krajcik, Adrian Jung, Andreas Hirsch, Winfried Neuhuber, and OliverZolk. Functionalization of carbon nanotubes enables non-covalent bindingand intracellular delivery of small interfering rna for efficient knock-downof genes. Biochem Biophys Res Commun, 369(2):595–602, May 2008. doi:10.1016/j.bbrc.2008.02.072.

A T Larregina, S C Watkins, G Erdos, L A Spencer, W J Storkus,D Beer Stolz, and L D Falo, Jr. Direct transfection and activation of hu-man cutaneous dendritic cells. Gene Ther, 8(8):608–17, Apr 2001. doi:10.1038/sj.gt.3301404.

74

Byung-Wan Lee, Hee-Young Chae, Tran Thi Ngoc Tuyen, Dongchul Kang,Hyun Ah Kim, Minhyung Lee, and Sung Hee Ihm. A comparison of non-viral vectors for gene delivery to pancreatic beta-cells: delivering a hypoxia-inducible vascular endothelial growth factor gene to rat islets. Int J Mol Med,23(6):757–62, Jun 2009.

Jie Liu, Zhiye Qiu, Shenqi Wang, Lei Zhou, and Shengmin Zhang. A mod-ified double-emulsion method for the preparation of daunorubicin-loadedpolymeric nanoparticle with enhanced in vitro anti-tumor activity. BiomedMater, 5(6):065002, Oct 2010. doi: 10.1088/1748-6041/5/6/065002.

Xianqiao Liu, Michael D Kaminski, Haitao Chen, Michael Torno, LaToyiaTaylor, and Axel J Rosengart. Synthesis and characterization of highly-magnetic biodegradable poly(d,l-lactide-co-glycolide) nanospheres. J Con-trol Release, 119(1):52–8, May 2007a. doi: 10.1016/j.jconrel.2006.11.031.

Zhuang Liu, Mark Winters, Mark Holodniy, and Hongjie Dai. sirna de-livery into human t cells and primary cells with carbon-nanotube trans-porters. Angew Chem Int Ed Engl, 46(12):2023–7, 2007b. doi: 10.1002/anie.200604295.

Holger Loessner and Siegfried Weiss. Bacteria-mediated dna transfer in genetherapy and vaccination. Expert Opin Biol Ther, 4(2):157–68, Feb 2004. doi:10.1517/14712598.4.2.157.

Dan Luo, Ernest Han, Nadya Belcheva, and W Mark Saltzman. A self-assembled, modular dna delivery system mediated by silica nanoparticles.J Control Release, 95(2):333–41, Mar 2004. doi: 10.1016/j.jconrel.2003.11.019.

Meredith A Mintzer and Eric E Simanek. Nonviral vectors for gene delivery.Chem Rev, 109(2):259–302, Feb 2009. doi: 10.1021/cr800409e.

S Moein Moghimi, Peter Symonds, J Clifford Murray, A Christy Hunter,Grazyna Debska, and Adam Szewczyk. A two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy. Mol Ther, 11(6):990–5, Jun 2005. doi: 10.1016/j.ymthe.2005.02.010.

Andreas Mörner, Iyadh Douagi, Mattias N E Forsell, Christopher Sundling,Pia Dosenovic, Sijy O’Dell, Barna Dey, Peter D Kwong, Gerald Voss, Rig-mor Thorstensson, John R Mascola, Richard T Wyatt, and Gunilla B Karls-son Hedestam. Human immunodeficiency virus type 1 env trimer immuniza-tion of macaques and impact of priming with viral vector or stabilized coreprotein. J Virol, 83(2):540–51, Jan 2009. doi: 10.1128/JVI.01102-08.

L Ngaboni Okassa, H Marchais, L Douziech-Eyrolles, S Cohen-Jonathan,M Soucé, P Dubois, and I Chourpa. Development and characteriza-

75

tion of sub-micron poly(d,l-lactide-co-glycolide) particles loaded with mag-netite/maghemite nanoparticles. Int J Pharm, 302(1-2):187–96, Sep 2005.doi: 10.1016/j.ijpharm.2005.06.024.

Sang Myoung Noh, Won-Ki Kim, Sun Jae Kim, Jung Mogg Kim, Kwang-Hyun Baek, and Yu-Kyoung Oh. Enhanced cellular delivery and transfectionefficiency of plasmid dna using positively charged biocompatible colloidalgold nanoparticles. Biochim Biophys Acta, 1770(5):747–52, May 2007. doi:10.1016/j.bbagen.2007.01.012.

S Ohkuma and B Poole. Fluorescence probe measurement of the intralyso-somal ph in living cells and the perturbation of ph by various agents. ProcNatl Acad Sci U S A, 75(7):3327–31, Jul 1978.

Davide Pantarotto, Ravi Singh, David McCarthy, Mathieu Erhardt, Jean-PaulBriand, Maurizio Prato, Kostas Kostarelos, and Alberto Bianco. Functional-ized carbon nanotubes for plasmid dna gene delivery. Angew Chem Int EdEngl, 43(39):5242–6, Oct 2004. doi: 10.1002/anie.200460437.

Jayanth Panyam, Wen-Zhong Zhou, Swayam Prabha, Sanjeeb K Sahoo, andVinod Labhasetwar. Rapid endo-lysosomal escape of poly(dl-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery. FASEBJ, 16(10):1217–26, Aug 2002. doi: 10.1096/fj.02-0088com.

Siddhesh D Patil, David G Rhodes, and Diane J Burgess. Dna-based ther-apeutics and dna delivery systems: a comprehensive review. AAPS J, 7(1):E61–77, 2005. doi: 10.1208/aapsj070109.

Dan Peer, Jeffrey M Karp, Seungpyo Hong, Omid C Farokhzad, RimonaMargalit, and Robert Langer. Nanocarriers as an emerging platform for can-cer therapy. Nat Nanotechnol, 2(12):751–60, Dec 2007. doi: 10.1038/nnano.2007.387.

Catarina Pinto Reis, Ronald J Neufeld, António J Ribeiro, and FranciscoVeiga. Nanoencapsulation i. methods for preparation of drug-loaded poly-meric nanoparticles. Nanomedicine, 2(1):8–21, Mar 2006. doi: 10.1016/j.nano.2005.12.003.

C W Pouton and L W Seymour. Key issues in non-viral gene delivery. AdvDrug Deliv Rev, 46(1-3):187–203, Mar 2001.

Swayam Prabha, Wen-Zhong Zhou, Jayanth Panyam, and Vinod Labhaset-war. Size-dependency of nanoparticle-mediated gene transfection: studieswith fractionated nanoparticles. Int J Pharm, 244(1-2):105–15, Sep 2002.

76

Chandrasekharan Prashant, Maity Dipak, Chang-Tong Yang, Kai-HsiangChuang, Ding Jun, and Si-Shen Feng. Superparamagnetic iron oxide–loaded poly(lactic acid)-d-alpha-tocopherol polyethylene glycol 1000 suc-cinate copolymer nanoparticles as mri contrast agent. Biomaterials, 31(21):5588–97, Jul 2010. doi: 10.1016/j.biomaterials.2010.03.070.

Indrajit Roy, Tymish Y Ohulchanskyy, Dhruba J Bharali, Haridas E Pu-davar, Ruth A Mistretta, Navjot Kaur, and Paras N Prasad. Optical track-ing of organically modified silica nanoparticles as dna carriers: a nonviral,nanomedicine approach for gene delivery. Proc Natl Acad Sci U S A, 102(2):279–84, Jan 2005. doi: 10.1073/pnas.0408039101.

Marika Ruponen, Paavo Honkakoski, Markku Tammi, and Arto Urtti. Cell-surface glycosaminoglycans inhibit cation-mediated gene transfer. J GeneMed, 6(4):405–14, Apr 2004. doi: 10.1002/jgm.522.

Sanjeeb K Sahoo, Jayanth Panyam, Swayam Prabha, and Vinod Lab-hasetwar. Residual polyvinyl alcohol associated with poly (d,l-lactide-co-glycolide) nanoparticles affects their physical properties and cellular uptake.J Control Release, 82(1):105–14, Jul 2002.

M Sameti, G Bohr, M N V Ravi Kumar, C Kneuer, U Bakowsky, M Nacken,H Schmidt, and C-M Lehr. Stabilisation by freeze-drying of cationicallymodified silica nanoparticles for gene delivery. Int J Pharm, 266(1-2):51–60, Nov 2003.

Kulmeet K Sandhu, Catherine M McIntosh, Joseph M Simard, Sallie WSmith, and Vincent M Rotello. Gold nanoparticle-mediated transfection ofmammalian cells. Bioconjug Chem, 13(1):3–6, 2002.

Mini Thomas and Alexander M Klibanov. Conjugation to gold nanoparticlesenhances polyethylenimine’s transfer of plasmid dna into mammalian cells.Proc Natl Acad Sci U S A, 100(16):9138–43, Aug 2003. doi: 10.1073/pnas.1233634100.

Eiichiro Uchimura, Shigeru Yamada, Lorenz Uebersax, Satoshi Fujita,Masato Miyake, and Jun Miyake. Method for reverse transfection using goldcolloid as a nano-scaffold. J Biosci Bioeng, 103(1):101–3, Jan 2007. doi:10.1263/jbb.103.101.

I M Verma and N Somia. Gene therapy – promises, problems and prospects.Nature, 389(6648):239–42, Sep 1997. doi: 10.1038/38410.

Wagner. Application of membrane-active peptides for nonviral gene delivery.Adv Drug Deliv Rev, 38(3):279–289, Aug 1999.

Shixia Wang, Swati Joshi, and Shan Lu. Delivery of dna to skin by particlebombardment. Methods Mol Biol, 245:185–96, 2004.

77

Liang WQ Wang WX, Chen HL. Study on polymethacrylate nanoparticlesas delivery system of antisense oligodeoxynucleotides. Yao Xue Xue Bao, 38(4):298–301, April 2003.

M Wilke, E Fortunati, M van den Broek, A T Hoogeveen, and B J Scholte.Efficacy of a peptide-based gene delivery system depends on mitotic activity.Gene Ther, 3(12):1133–42, Dec 1996.

R S Williams, S A Johnston, M Riedy, M J DeVit, S G McElligott, and J CSanford. Introduction of foreign genes into tissues of living mice by dna-coated microprojectiles. Proc Natl Acad Sci U S A, 88(7):2726–30, Apr1991.

M A Wolfert, P R Dash, O Nazarova, D Oupicky, L W Seymour, S Smart,J Strohalm, and K Ulbrich. Polyelectrolyte vectors for gene delivery: in-fluence of cationic polymer on biophysical properties of complexes formedwith dna. Bioconjug Chem, 10(6):993–1004, 1999.

Yongxin Yang, Zhiwen Zhang, Lingli Chen, Wangwen Gu, and Yaping Li.Galactosylated poly(2-(2-aminoethyoxy)ethoxy)phosphazene/dna complexnanoparticles: in vitro and in vivo evaluation for gene delivery. Biomacro-molecules, 11(4):927–33, Apr 2010. doi: 10.1021/bm901346m.

Xuan Zeng, Yun-Xia Sun, Wei Qu, Xian-Zheng Zhang, and Ren-Xi Zhuo.Biotinylated transferrin/avidin/biotinylated disulfide containing pei biocon-jugates mediated p53 gene delivery system for tumor targeted transfection.Biomaterials, 31(17):4771–80, Jun 2010. doi: 10.1016/j.biomaterials.2010.02.039.

Haiyuan Zhang, Moo-Yeal Lee, Michael G Hogg, Jonathan S Dordick, andSusan T Sharfstein. Gene delivery in three-dimensional cell cultures by su-perparamagnetic nanoparticles. ACS Nano, 4(8):4733–43, Aug 2010. doi:10.1021/nn9018812.

Yang Zhao, Yang Lu, Yan Hu, Jian-Ping Li, Liang Dong, Li-Ning Lin,and Shu-Hong Yu. Synthesis of superparamagnetic caco(3) mesocrys-tals for multistage delivery in cancer therapy. Small, Sep 2010. doi:10.1002/smll.201000903.

78

a novel design of gene therapy carriers - ph …...a novel design of gene therapy carriers - ph...

Documents