ABSTRACT

LIU, YANLI. Nanoparticle-based Delivery Vectors: Design, Preparation, Characterization,

Cellular Internalization and Nuclear Targeting. (Under the Direction of Daniel L. Feldheim.)

The use of nucleic acids as therapeutics is an attractive strategy for genetic disease

treatment. The major hurdle is the low cellular delivery efficacy. Virus-mimetic nanoparticle-

based vectors combine several different components (targeting peptides/stabilizer and

therapeutics) onto a single nanoparticle and thus capable of traversing cellular membranes,

escaping degradative pathway and targeting the nucleus of the cell.

One challenge in the synthesis of nanoparticle vectors is the stability because the

incorporation of cell-targeting peptides onto the nanoparticle surface frequently compromises

nanoparticle stability in biological media. The assembly of PEG and mixed peptide/PEG

monolayers on gold nanoparticle surfaces were described herein. The stability of the

resulting bioconjugates in high ionic strength media was characterized as a function of

nanoparticle size, PEG length, and monolayer composition. In total, three different thiol-

modified PEGs (Average MW 900, 1500 and 5000 g mol-1), four particle diameters (10 nm,

20 nm, 30 nm and 60 nm), and two cell-targeting peptides were explored. It was found that

nanoparticle stability increased with increasing PEG length, decreasing nanoparticle

diameter, and increasing PEG mol fraction. The order of assembly also played a role in

nanoparticle stability. Mixed monolayers prepared via the sequential addition of PEG

followed by peptide were more stable than particles prepared via simultaneous co-adsorption.

Finally, the ability of nanoparticles modified with mixed PEG/RME peptide monolayers to

target the cytoplasm of HeLa cells was quantified using Inductively Coupled Plasma Optical

Emission Spectrometry (ICP-OES). Although it was anticipated that the MW 5000 g mol-1
PEG would sterically block peptides from access to the cell membrane compared to the MW

900 PEG, nanoparticles modified with mixed peptide/PEG 5000 monolayers were

internalized as efficiently as nanoparticles containing mixed peptide/PEG 900 monolayers.

The second strategy to build nanoparticle-based delivery vectors was to prepare

(antisense oligonucleotide + peptides)-coated nanoparticles via streptavidin(SA)-biotin

linkage. The conjugates were characterized by gel electrophoresis, UV-vis absorption

spectroscopy, dynamic light scattering (DLS) and fluorescence spectroscopy. The vectors

were delivered into pLuc HeLa cells and analyzed by luciferase assay system. By using

alternate gene splicing as a positive readout assay, the bioactivities of the delivery vectors

were studied. Five peptides, two nanoparticles (gold nanoparticles and quantum dots), and

two oligonucleotides (ON705 and PNA) were examined. Findings were reported herein: (1)

Antisense oligonucleotides modified with nanoparticles were bio-functional to pre-mRNA

in the nucleus of the cells and the bio-availability was independent of nanoparticles,

therapeutics and bioconjugate strategies; (2) The nanoparticles improve the nuclear

targeting efficiency of oligonucleotides by up to 5 fold with Lipofectamine (LF) compared

with free oligonucleotides with LF.

These studies provide new insights into the design, possibilities and limitations of

the biological applications of multifunctional nanoparticle vectors.

 NANOPARTICLE-BASED DELIVERY VECTORS:
DESIGN, PREPARATION, CHARACTERIZATION,
CELLULAR INTERNALIZATION AND NUCLEAR TARGETING

by

YANLI LIU

A dissertation submitted to the Graduate Faculty of

North Carolina State University
In partial fulfillment of the
Requirements for the degree of
Doctor of Philosophy

Chemistry

North Carolina State University, Raleigh, NC

April. 2007

Approved by:

Dr. Daniel L. Feldheim Dr. Edmond F. Bowden

Advisory Committee, Chairman Advisory Committee, member

Dr. Christian Melander Dr. Lin He

Advisory Committee, member Advisory Committee, member
 DEDICATION

This work is dedicated to my family.

ii
 BIOGRAPHY

Yanli Liu was born in Shan Dong Province in China to Father Honglin Liu and

Mother Cuiting Yu with an older sister Yanmei Liu. She got both her bachelor degree in

pharmaceutical engineering and her master degree in medicinal chemistry in East China

University of Science and Technology in Shanghai, China during 1995-2002. She then

worked in a pharmaceutical company in Shanghai as a research fellow for about six months.

In the summer of 2002, she came to the United States, starting a 5-year graduate study in NC

State University-Chemistry Department.

She is married to Dazhong Fan with a lovely daughter, Crystal, who was born on

September 30, 2006.

iii
 ACKNOWLEDGMENTS

First, I would like to thank my research advisor, professor Dan Feldheim. He had

been very supportive and patient with me during the past five years. No matter how busy he

was, he was always willing to sit down with me as a good friend and a wise research mentor.

I am grateful for his support and guidance.

Second, I would like to thank professor Stefan Franzen. He has wide knowledge in

both chemistry and biology. Talking to him benefits me a lot in many different ways.

Then, I would like to thank all my friends from both Feldheim and Franzens’ group.

It was them who taught me various lab techniques, discussed experiments and data with me

on a daily basis. Without them, it would not be so much fun in the lab.

At last but not the least, I would like to thank my family for their endless love and

continuous support. I owe much to my parents for their unconditional love, support and

always believing in me. My husband, Dazhong, is always there for me no matter what.

Crystal, my lovely daughter, brings me so much happiness.

Without all of you, I would not become the person who I am today. For that, I would

like to thank everyone for being here for me.

iv
 TABLE OF CONTENTS

List of Figures ………………………………………………………………………………...x

List of Tables ……………………………………………………………………………… xiii

Chapter 1 Cellular Internalization and Nuclear Targeting of Oligonucleotides with

Nanoparticle-based Synthetic Vectors...................................................................................... 1

1.1 Background/ Introduction ............................................................................................... 2

1.2 Non-viral delivery vectors .............................................................................................. 3

1.2.1 Inorganic chemicals ................................................................................................. 3

1.2.2 Cationic lipids .......................................................................................................... 4

1.2.3 Cationic polymers .................................................................................................... 5

1.2.4 Metal nanoparticles.................................................................................................. 5

1.3 Viruses and viral delivery vectors................................................................................... 7

1.4 Proposed hybrid nanoparticle delivery vectors............................................................... 9

1.4.1 Carrier .................................................................................................................... 11

1.4.1.1 Gold nanoparticles .......................................................................................... 12

1.4.1.2 Nanocrystals, Quantum dots (QDs) ................................................................ 12

1.4.2 Cell-targeting peptides ........................................................................................... 13

1.4.3 Therapeutics: antisense oligonucleotides............................................................... 15

1.5 Strategies to prepare nanoparticle-based delivery vectors conjugates.......................... 17

1.6 Size selection ................................................................................................................ 20

1.7 Intracellular transport and processing of synthetic oligonucleotide vectors................. 20

v
 1.7.1 Cellular binding and internalization....................................................................... 21

1.7.2 Escape from the degradative pathway ................................................................... 23

1.7.3 Cytosolic trafficking .............................................................................................. 23

1.7.4 Nuclear delivery..................................................................................................... 24

1.8 The splicing assay as a positive readout for nuclear targeting...................................... 25

1.9 Summary ....................................................................................................................... 27

1.10 References................................................................................................................... 28

Chapter 2 Gold Nanoparticles: Synthesis, Functionalization, Stability and Biological

Applications ............................................................................................................................ 34

2.1 A brief historic introduction.......................................................................................... 35

2.2 Chemical synthesis of colloidal gold ............................................................................ 36

2.2.1 Citrate reduction in aqueous solutions................................................................... 37

2.2.2 Reduction from non-aqueous solutions ................................................................. 38

2.3 Strategies for functionalization of gold nanoparticles .................................................. 39

2.3.1. Formation of nanoparticles in the presence of thiols ............................................ 39

2.3.2. Ligand exchange methods..................................................................................... 41

2.3.3. Covalent modification........................................................................................... 43

2.3.4 Electrostatic binding .............................................................................................. 44

2.4 Stability of colloidal gold.............................................................................................. 44

2.4.1 Critical Coagulation Concentration (CCC)............................................................ 45

2.4.2 Van der Waals forces ............................................................................................. 46

2.4.3 Electrostatic stabilization ....................................................................................... 46

2.4.4 Steric stabilization.................................................................................................. 47

vi
 2.4.5 Electrosteric stabilization....................................................................................... 49

2.4.6 Polymeric stabilization and flocculation................................................................ 50

2.4.6.1 Effects of adsorbed polymer ........................................................................... 50

2.4.6.2 Effects of free polymer ................................................................................... 52

2.5 Properties and applications of gold nanoparticles ........................................................ 53

2.5.1 Physical-chemical properties and the use as a platform to display multiple

polyvalent ligands ........................................................................................................... 53

2.5.2 Optical properties of gold nanoparticles and their biological applications........... 54

2.6 Summary ....................................................................................................................... 57

2.7 References..................................................................................................................... 59

Chapter 3 Synthesis, Stability, and Cellular Internalization of Gold Nanoparticles Containing

Mixed Peptide-Poly(ethylene glycol) Monolayers ................................................................. 65

3.1 Introduction................................................................................................................... 66

3.2 Experimental Methods .................................................................................................. 67

3.2.1 Reagents and instrumental ..................................................................................... 67

3.2.2 Synthesis of PEG-SH molecules............................................................................ 68

3.2.3 Preparation of pegylated gold nanoparticles.......................................................... 70

3.2.4 Preparation of gold nanoparticles with mixed monolayers of PEG and peptides.. 70

3.2.5 Determination of critical coagulation concentrations (CCC) ................................ 71

3.2.6 Quantifying peptide adsorption onto gold nanoparticles by fluorescence

spectroscopy.................................................................................................................... 72

3.2.7 Characterization of protein non-specific binding using fluorescence spectroscopy

......................................................................................................................................... 72

vii
 3.2.8 Cell treatment with gold nanoparticles .................................................................. 73

3.2.9 Sample preparation for inductively coupled plasma optical emission spectroscopy

(ICP-OES)....................................................................................................................... 74

3.3 Results........................................................................................................................... 75

3.3.1 Assembly of PEG-coated gold nanoparticles ........................................................ 76

3.3.2 Non-specific binding of proteins to PEG-modified gold nanoparticles................. 83

3.3.3 Assembly of mixed PEG/Peptide monolayers on gold nanoparticles. .................. 84

3.4 Discussion ..................................................................................................................... 93

3.5 Conclusions................................................................................................................. 100

3.6 References................................................................................................................... 101

Chapter 4 Antisense Oligonucleotide Modified Nanoparticles for Up-regulation of Luciferase

Gene Expression ................................................................................................................... 104

4.1 Introduction................................................................................................................. 105

4.2 Materials and Instrumentation .................................................................................... 106

4.3 Experimental procedures ............................................................................................ 108

4.3.1 Preparation of streptavidin (SA) coated gold nanoparticles ................................ 108

4.3.2 Determination of surface coverage of Streptavidin (SA) on gold nanoparticles . 108

4.3.3 Annealing and confirmation of Peptide Nucleic Acid (PNA) to Leash............... 109

4.3.4 Preparation of leash-PNA duplex coated gold nanoparticles/Quantum dots (QDs)

....................................................................................................................................... 109

4.3.5 Preparation of peptide-modified QDs.................................................................. 110

4.3.6 Preparation of ON705-SA-gold nanoparticles..................................................... 110

4.3.7 Preparation of (ON705 + peptides)-SA-gold nanoparticles ................................ 110

viii
 4.3.8 Preparation of ON705-thiol (S)- gold nanoparticles............................................ 111

4.3.9 Determination of surface coverage of oligonucleotides on particle surface........ 111

4.3.10 Determination of critical coagulation concentrations (CCC) ............................ 112

4.3.11 Nanoparticle transfection with pLuc HeLa cells ............................................... 112

4.4 Results......................................................................................................................... 113

4.4.1 Assembly and characterization of (peptides+ oligonucleotides)-coated particles113

4.4.2 Antisense activity of nanoparticles examined by luciferase assay system .......... 116

4.4.2.1 Relative antisense activity of gold nanoparticles modified solely with SA-

oligonucleotides ........................................................................................................ 117

4.4.2.2 Relative antisense activity of gold nanoparticles modified with both peptide

and oligonucleotides ................................................................................................. 117

4.4.3 Relative antisense activity of QDs modified peptide and ON705/PNA .............. 122

4.4.4 The bioavailability of antisense oligonucleotides modified by nanoparticles ..... 122

4.5. Discussion .................................................................................................................. 125

4.6 Conclusion .................................................................................................................. 128

4.7 References................................................................................................................... 129

ix
 LIST OF FIGURES

Figure 1.1 Viruses enter cells.................................................................................................... 7

Figure 1.2 Illustration of a nanoparticle-based delivery vector .............................................. 10

Figure 1.3 Schematic of the overall structure of SA-QD conjugate. ...................................... 12

Figure 1.4 Chemical structure of naturally occurring and modified oligonucleotides. .......... 17

Figure 1.5 Covalent coupling of PEG and peptides to a nanoparticle surface. ...................... 18

Figure 1.6 The synthesis of peptide-modified gold nanoparticle conjugates for cellular

targeting. ......................................................................................................................... 19

Figure 1.7 The synthesis of (peptide+oligonucleotide)-modified gold nanoparticle conjugates

via streptavidin-biotin interaction. .................................................................................. 20

Figure 1.8 The splicing assay as a positive readout for nuclear targeting. ............................. 27

Figure 2.1 Formation of gold nanoparticles by reduction of HAuCl4 in the presence of thiols.

......................................................................................................................................... 40

Figure 2.2 General scheme for the ligand-exchange reaction between alkanethiol-gold

nanoparticles of the Brust type and various functionalized thiols. ................................. 41

Figure 2.3 Surface functionalization of gold nanoparticles with coumarin-PEG-thiol through

ligand exchange. ............................................................................................................. 43

Figure 2.4 Schematic representation of steric stabilization of colloid particles. .................... 48

Figure 2.5 Plot of potential energy V(d) vs interparticle distance (d) for electrostatic

stabilization (A) steric stabilization (B).......................................................................... 49

Figure 2.6 Effects of polymer chains on colloidal dispersions............................................... 50

Figure 2.7 Bridging flocculation of colloidal particles by polymeric flocculants. ................. 51

Figure 2.8 Schematic representation of the origin of depletion flocculation.......................... 52

x
Figure 2.9 Gold nanoparticle-based DNA biosensor as a molecular beacon. ........................ 57

Figure 3.1 Synthesis of PEG 900 (n = 20) and PEG 1500 (n = 34) molecules. ..................... 68

Figure 3.2 1% Agarose gel electrophoresis of 20 nm diameter gold nanoparticles following

the addition of different ratios of PEG 5000 (left) and PEG 900 (right).. ...................... 77

Figure 3.3 Two data sets measuring the z-potential of PEG 5000 and PEG 900 modified 20

nm diameter gold nanoparticles. ..................................................................................... 78

Figure 3.4 UV-visible absorption spectra of 20 nm diameter citrate-stablized gold

nanoparticles incubated with varying concentrations of NaCl. ...................................... 80

Figure 3.5 UV-vis absorption spectra of PEG 5000 stabilized gold nanoparticles (20 nm in

diameter) in 500 mM NaCl solution (final concentration). ............................................ 80

Figure 3.6 CCC of pegylated gold nanoparticles (10nm in diameter).................................. 81

Figure 3.7 UV-visible absorption spectra of 10 nm diameter gold nanoparticles modified with

PEG 5000 and PEG 5000 + RME peptide (left). Agarose gel electrophoresis of gold

nanoparticles (right) ........................................................................................................ 85

Figure 3.8 Adsorption isotherm of rhodamine-labeled RME peptide adsorbed onto 10 nm

diameter gold nanoparticles.. .......................................................................................... 86

Figure 3.9 Adsorption isotherms of rhodamine-labeled RME peptide adsorbed onto 20 nm

diameter gold nanoparticles ............................................................................................ 87

Figure 3.10 Nonspecific binding of BSA to (PEG 5000 + RME peptide)-coated 20 nm

diameter gold nanoparticles (top) and (PEG 900 + RME peptide)-coated gold

nanoparticles (bottom). ................................................................................................... 90

xi
Figure 3.11 ICP-OES analysis of HeLa cells following 6 hours incubation with media

containing 10 nm diameter gold particles modified with mixed PEG/RME monolayers

......................................................................................................................................... 92

Figure 3.12 side and top projections of Gaussian chain on a surface ..................................... 97

Figure 3.13 Circular projection of Gaussian chain ................................................................. 98

Figure 4.1 1% Agarose gel electrophoresis of ON705-SA-gold nanoparticles. ................... 114

Figure 4.2 1% Agarose gel electrophoresis of modified 10 nm diameter gold nanoparticles.

....................................................................................................................................... 114

Figure 4.3 Relative antisense activities of modified gold nanoparticles .............................. 118

Figure 4.4 Representative examples of particle formulations with (peptide+

oligonucleotide) ............................................................................................................ 119

Figure 4.5 Relative antisense activity of gold nanoparticles modified with peptide and

ON705 in the absence of LF. ........................................................................................ 119

Figure 4.6 Relative antisense activity of gold nanoparticles modified with peptide and

ON705 in the presence of LF........................................................................................ 120

Figure 4.7 Relative antisense activity of different amounts of ON705-gold and ON705 only

with 8 µg of LF in pLuc 705 HeLa cells....................................................................... 121

Figure 4.8 Relative antisense activity of ON705-gold and ON705 only with a fixed

concentration of ON705 (0.3 µM) in pLuc 705 HeLa cells.......................................... 122

Figure 4.9 Comparison of relative antisense activity of nanoparticles with different linkers in

the presence of LF......................................................................................................... 123

Figure 4.10 Comparison of relative antisense activity of ON705 modified with different type

of nanoparticles in the presence of LF.......................................................................... 124

xii
 LIST OF TABLES

Table 1.1 Comparison of viral vectors, non-viral vectors and nanoparticle vectors ........ 11

Table 3.1 Peptides used for assembling gold nanoparticle conjugates............................ 68

Table 3.2 Average molecular weights and ethylene glycol (EG) units of the PEGs

investigated in this study........................................................................................... 76

Table 3.3 Dynamic Light Scattering (DLS) of 20 nm diameter gold particles modified with

the indicated monolayer............................................................................................ 79

Table 3.4 Minimum number of PEG molecules that must be added into the reaction mixture

per gold nanoparticle in order to form a stable gold sol in 1 M NaCl. ..................... 83

Table 3.5 Nonspecific binding of proteins to PEG 5000-modified gold nanoparticles.... 84

Table 3.6 CCC of 20 nm diameter gold nanoparticles modified with mixed monolayers of

PEG 5000 and the NLS peptide. ............................................................................... 88

Table 3.7 CCC of 10 nm diameter gold nanoparticles modified with mixed monolayers of

PEG 900 and the RME peptide................................................................................. 89

Table 3.8 Nonspecific binding of protein BSA to 20 nm diameter gold nanoparticles

modified with the indicated monolayer. ................................................................... 91

Table 4.1 Peptides used in this study .............................................................................. 107

Table 4.2 Oligonucleotides used in this study ................................................................ 107

Table 4.3 Dynamic Light Scattering (DLS) of 10 nm diameter gold particles modified with

the indicated molecules........................................................................................... 115

Table 4.4 CCC of 10 nm diameter gold nanoparticle modified with SA and ON705.... 116

Table 4.5 Chemical linkers used in the study ................................................................. 123

Table 4.6 Nanoparticles used in the study ...................................................................... 124

xiii
Chapter 1 Cellular Internalization and Nuclear Targeting of

Oligonucleotides with Nanoparticle-based Synthetic Vectors

1
 This chapter will start with a background introduction of cellular delivery of nucleic

acids. Then, non-viral delivery vectors will be discussed followed the discussions of viruses

and viral vectors. Inspired by the chemical structures of viruses, artificial virus delivery

vector-nanoparticle-based delivery system will be proposed, which will have applications in

the delivery of oligonucleotides (antisense oligonucleotides and short interference RNA) as

well as potentially for small molecules such as cancer drugs. Major components of such a

delivery system including carrier, targeting signal and therapeutics will be individually

addressed. More over, bioconjugate strategies, the size selection of nanoparticles, possible

cellular processes of the vectors and a bioactivity assay to test the functionality of the

delivery vectors will be discussed.

1.1 Background/ Introduction

As many diseases (Breast cancer, Melanoma, Hemophilia, Alzheimer’s disease and

Parkinson’s disease) have a genetic basis, the use of nucleic acids (that is, DNA, antisense

oligonucleotides and small interfering RNAs (siRNA)) as therapeutic agents to correct

missing genes, replace defective genes or up-regulate aberrant gene expression by targeting a

number of intracellular targets is an attractive strategy for genetic disease treatment. Several

approaches, including gene therapy, antisense therapy, siRNA silencing and DNA

vaccination, have become novel ways to fight disease. For example, triple helix and

transcription factor decoy are two methods that have been applied to gene down-regulation1-
4
. More recently RNA interference has proven a useful method using short 21-22 nucleotide-

long double-stranded RNA molecules that silence genes by degrading mRNA in the RISC

complex5, 6. Antisense oligonucleotides can be used not only in the conventional way, which

2
is by down-regulation of gene expression via RNase H-promoted degradation of targeted

mRNA, but also for modification of the splicing patterns of genes7-10. With the promise of

specificity and low toxicity, it is anticipated that these technologies will have an enormous

impact on medicine and biotechnology. To date, more than 1,000 different gene-therapy

clinical trials for the treatment of many different diseases are in progress worldwide. Several

products have reached phase II or III clinical trials11. The first commercial gene medicine, a

recombinant adenoviral vector carrying a p53 gene for head and neck squamous cell

carcinoma, was recently approved in China12.

Notwithstanding this large number of ongoing trials, success with gene therapy has

been limited and has not yet resulted in approval of a gene therapy product by the US FDA.

This can be ascribed to difficulties related to the effective introduction of nucleic acids into

target cells, which requires the use of sophisticated delivery systems. Generally, for the

introduction of nucleic acid, two different types of delivery systems can be distinguished:

those that are derived from viruses, called viral vectors, such as retroviral vectors,

recombinant adenoviral vectors and adeno-associated virus (AAV) vectors, and those that

are manmade called non-viral vectors including cationic lipids and polymers, dendrimers,

liposomes, and nanoparticles13-18.

1.2 Non-viral delivery vectors

1.2.1 Inorganic chemicals

Naked DNA plasmids are rapidly degraded by plasma nuclease, which results in a

low transfection (the introduction of foreign materials into eukaryotic cells) yield. A popular

3
pathway to transport DNA/RNA inside cells is to use carrier molecules. In the early 1970s,

calcium phosphate was employed to transfect DNA19. Calcium phosphate coprecipitated with

DNA is one of the most commonly used non-viral vectors. However, the transfection of

calcium phosphates is normally insufficient due to the dependence on particle size under

certain precipitation conditions, the protection of DNA from enzyme degradation and the

type of cells.

1.2.2 Cationic lipids

Cationic lipids are amphiphilic molecules that comprise a polar (cationic) head linked

to a hydrophobic tail via a spacer. They can self-assemble by co-operative hydrophobic

intermolecular binding at a certain concentration, forming cationic liposomes. The lipids can

then bind and compact nucleic acid by electrostatic interaction between the positively

charged polar heads of the lipids and the negatively charged phosphate groups of the DNA,

forming cationic lipid/DNA complexes, a.k.a. lipoplexes13, 20. The nucleic acid is protected

from the degradation of nucleases and is able to reach the desired site of the cells. Cationic

lipids especially facilitate the transfection during the early stage of the intracellular process

by condensing the DNA and binding with cell membranes. Cationic lipids offer greater ease

of use and efficiency with low toxicity. However, cationic lipids become cytotoxic, usually

resulting in 30-40% loss in viability beyond certain charge ratio (lipid/DNA=3:1)20.

Regarding the lipoplex-mediated gene delivery, several cationic lipids were approved for

clinical trials, such as DC-Chol for breast an ovarian cancer21and DMRIE for human basal

cell carcinoma22.

4
1.2.3 Cationic polymers

With regard to polymers for gene/drug delivery, major attention is paid to cationic

polymers , which are able to condense large genes into small structures and to mask the

negative DNA charges, necessities for transfecting most types of cells. Complexes of cationic

polymers with DNA are called polyplexes and their production is regulated by ionic

interactions. About four decades ago, it was discovered that coating DNA with

Diethylaminoethyl-dextran (DEAE-dextran) would allow DNA to transit across the cell

membranes. Currently, commonly used cationic polymers include poly(L-lysine) (PLL),

polyethylenimines (PEI), polyamidoamine dendrimers(PAMAM), chitosan23 . Among them,

PEI and PAMAM are the most frequently used because of the excellent transfection

efficiency. It has been shown that (PAMAM) (< 5 nm) can selectively target tumor cells
17, 24
when loaded with an anticancer drug . The main disadvantage of these polymers are the

pronounced toxicity.

1.2.4 Metal nanoparticles

Metal nanoparticles generally possess versatile properties suitable for cellular

delivery, including wide availability, rich functionality, good biocompatibility, potential

capability of targeted delivery (e.g. selectively destroying cancer cells but sparing normal

tissues) and controlled release of therapeutics. Therefore, many metal materials, such as gold,

magnetic nanoparticles and nanoshells, have been extensively studied.

The idea of using magnetic particles as therapeutic drug carriers dates back to the late

1970s25. Magnetic particles may be functionalized with carboxyl groups, amines, biotin,

streptavidin, antibodies, and others26, 27. Magnetic particles may also be embedded within

5
hydrogels, carry a therapeutic agent and release it upon heating, or the particles themselves

may be used for hyperthermia application25. Magnetic nanoparticle-based targeting could

reduce or eliminate the side effects of chemotherapy drugs by reducing their systemic

distribution as well as by administering lower but more accurately targeted doses of the

cytotoxic compounds used in the treatment.

Gold nanoparticles can also be modified with suitable biomolecules to target certain

compartments of cells. For example, it has been shown in a preclinical mouse model that

when TNF-α was systemically delivered as nanogold-conjugates, they exhibited reduced

toxicity and better efficacy compared to TNF-α alone16. The Mirkin group has recently

showed the use of oligonucleotide-functionalized gold nanoparticles as new antisense

agents to target the cytoplasm of cells28. These oligonucleotide-modified nanoparticles have

affinity constants for complementary nucleic acids that are higher than their unmodified

oligonucleotide counterparts and they are less susceptible to degradation by nuclease

activity. Therefore, those particles can translocate oligonucleotides at a higher effective

concentration than conventional transfection agents.

Gold nanoshells are a new class of nanoparticles with highly tunable properties.

They are concentric sphere nanoparticles consisting of a dielectric (typically gold sulfide or

silica) core and a metal (gold) shell. Potential biomedical applications of the nanoshells

include immunoassays, modulated drug delivery, photothermal cancer therapy and imaging

contrast agents29-31. For example, composites of thermally sensitive hydrogels and optically

active nanoparticles have been developed for drug delivery32. In this method, metal

nanoparticles release their drugs that are held in the hydrogel matrix such as methylene blue

6
 and proteins of varying molecular weight, into the targeted location using an external

exciting source like an infrared light or a magnetic field32.

A possible problem is that the excretion of inorganic nanoparticles and their

accumulation in the cell may harm the cell growth. Due to the high chemical stability,

inorganic nanoparticles cannot be dissolved easily in the cell; and excretion is difficult due to

their relatively large size.

1.3 Viruses and viral delivery vectors

Owing to the multiple components (lipid, proteins and the genome (DNA or RNA))

and evolutionarily optimized properties, viruses have evolved the chemistry to bind to cell

surfaces, cross cellular membranes, hijack intracellular transport systems and subsequently

deliver their genomes into the appropriate subcellular compartment (for example, cytosol or

nucleus).

Figure 1.1 Viruses enter cells

7
 Ports of cell entry include sugar receptors, hormone receptors and ion channels. Ion

channels are usually too small for large molecules. The sugar and hormone receptors are

linked to endocytosis (the cellular uptake of molecules by localized regions of the plasma

membrane that surround the substance and pinch off to form an intracellular vesicle). For the

adenovirus, the long fibrous protein acting as the cell-targeting signal gains cell entry by

receptor-mediated endocytosis. Once inside, the virus releases an enzyme to rupture the wall

of the vesicles. Thus, the viral capsid (repeating hexon and penton proteins) is released to the

cytoplasm. AIDS HIV virus binds to cell receptors but does not activate endocytosis. The

viral envelope fuses with the cell membrane, releasing the capsid directly into the cytoplasm

without the formation of a vesicle. In either case, once the capsid is free in the cytoplasm, it

breaks open to release the viral chromosome.

When an adenovirus enters the cell, the nuclear localization sequences (NLS) on the

partially fragmented capsid bind to a nuclear pore and mediate extrusion of the nucleic acids

via the nuclear pore complex (NPC), after which the chromosome moves into the nucleus,

where it is replicated and transcribed. When a retrovirus enters the cell, the capsid core

breaks open to release the RNA genome into the cytoplasm, which is quickly converted to

DNA by reverse transcriptase. The viral DNA moves into the nucleus, where it is integrated

into the genome33.

Given their ability to enter cells, viruses would appear to be excellent candidates for

gene delivery vectors.

Adenovirus type 2 (AD-2) and a retrovirus called murine leukemia virus (MuLV)

have been used in more than 90 percent of all gene therapy trials to date. However, many

challenges still exist. The right dosage of AD-2 is crucial, where AD-2 is injected directly in

8
a trial treatment of liver ailment. If the amount is too low, few cells will take up the virus, so

the expression of the therapeutics will be insufficient to treat the disease. If the amount is too

high, viral particles will spill out and infect other cells. In extreme cases, this will cause the

destruction of entire organs and death of the patient. Further, AD vectors are inefficient at

infecting some cells, and they tend to activate an antivector immune response.

Alternatively, retroviruses have the ability of infecting a wide variety of cells. They

have the potential of integration into host genomes. The problems with current retroviral

systems include inability to produce high titers; immunogenic problems34.

Overall, major restrictions of viral vectors, such as limited DNA-carrying capacity,

lack of target-cell specificity, immunogenicity and, for some viral vectors, insertional

mutagenesis, limit their use in clinical settings.

Nevertheless, viruses provide a set of design features that are mimicked in the

development of nonviral vectors: condensation of DNA/RNA into nanoparticles, protection

from nuclease degradation, binding to cells, delivery into the cytoplasm and (for nondividing

cells) the nucleus, and unpacking to release the nucleic acids.

1.4 Proposed hybrid nanoparticle delivery vectors

The general idea of the synthetic vector is to deliver nucleic acid (DNA and RNA) as

well as small molecules like drugs into certain subcellular compartments for cancer therapy.

The ultimate goal is to prepare an artificial hybrid vector that equals viral counterparts in

terms of transfection efficiency, but is safe to use. The vector should (1) remain stable during

its transport through the cellular barriers (2) be small enough (3) traverse several biological

barriers from the cells and the environments (4) deliver the oligonucleotides to the target

9
subcellular compartment (i.e. epithelium, mitochondria, cytosol, nucleus) (4) protect the

carried molecules from degradation. Obviously, such a vector is difficult to obtain because it

requires the combination of several different and often counteracting components in a single

particle. To unite all the required components onto a single nanoparticle without losing any

of the functionalities of the individual components is the major challenge in the design of

hybrid vectors.

The approach described here involves the use of naturally occurring or synthetic

peptides, combined with short nucleic acids on a nanometer-sized particle. The vector

consists of several components: carrier, targeting signal, therapeutics and/or stabilizer if

necessary (Figure 1.2), which will be individually addressed in the next few sections of this

chapter.

Therapeutics

t icle
a n opar
N Targeting Signals

Stabilizer

Figure 1.2 Illustration of a nanoparticle-based delivery vector

The concept of a hybrid nanoparticle delivery vector with multifunctional ligands

arises by analogy with a virus. However, there is a profound difference between a virus and a

nanoparticle vector. The goal of viruses is to deliver DNA to the nucleus in order to replicate

10
and create new virions. The nanoparticle vector mimics the viral functions that permit

translocation of genetic material into the nucleus without infections. While viruses are

extremely well adapted to carrying and delivering genomic DNA or RNA, they are not well

suited for the delivery of drugs and oligonucleotides. Non-viral vectors such as liposomes,

polymers, dendrimers etc. have been adapted to gene delivery and drug delivery in general.

Non-viral vectors have a range of possible payloads in a drug delivery strategy. However,

nanoparticle delivery vectors have a number of advantages over the general class of non-viral

delivery vectors as shown in Table 1.1.

Table 1.1 Comparison of viral vectors, non-viral vectors and nanoparticle vectors
Viral vectors Non-viral vectors Nanoparticle vector
Efficiency High Low Can be high
Function determined by viral
Synthesis and Hard to incorporate Easy to incorporate different
structures and not easily
Modification multiple functions functions on a single particle
modified
Immunogenicity Elicit strong immune response Can be controlled Can be controlled
Individual
Size restricted dendrimers (<2 nm) Size tunable from 1 nm to 200
Size
(30 – 100 nm) Polymers (> 50 nm) nm
Liposome (> 20 nm)

1.4.1 Carrier

Appropriate choice of a carrier is critical to the design of a hybrid nanoparticle vector.

In my research, I mainly focused on two nanoparticles: gold nanoparticles with a diameter

range from 5 nm to 30 nm (section 1.4.1.1 and chapter 2) and nanocrystals, quantum dots

(section 1.4.1.2).

11
1.4.1.1 Gold nanoparticles

The ease of synthesis and functionalization, unique optical properties and

biocompatibility make gold nanoparticles excellent candidate as the carrier for intracellular

delivery. Synthesis, modification, optical properties and their biological applications will be

discussed in chapter 2.

1.4.1.2 Nanocrystals, Quantum dots (QDs)

QDs possess unique luminescent properties. Their fluorescence emission is very

stable compared to regular organic fluorophores and tunable by varying particle size or

chemical composition. Moreover, such stable inorganic nanoparticles can be conjugated with

various biomolecules, such as proteins, peptide and nucleic acids. There have been numerous

publications about QDs as novel intravascular probes for both imaging and drug delivery35-38.

Naik et al. showed that QDs modified with a NLS peptide and an apoptotic trigger could

induce mitochondrial-mediated cell death39.

Streptavidin (SA) coated nanocrystals (CdSe@ZnS), QD605, was commercially

available and was used in the study. The structure was illustrated in figure 1.3.

Figure 1.3 Schematic of the overall structure of SA-QD conjugate.

12
1.4.2 Cell-targeting peptides

Different types of targeting ligands have been used for cellular internalization and

cellular targeting including peptides, antibodies, sugar and vitamins40-42. Our approach to

improving the efficacy of delivery devices is the addition of short virus-mimetic targeting

peptides into the formulation of the delivery vectors. Such peptides are used because of prior

observations that the functionally active portions of many viral proteins consist of relatively

small sequences, typically of around 10-20 amino acids. Specific sequences of interest for

cell delivery include peptide ligands for cell recognition, receptor-mediated uptake,

endosomal release, and peptides that facilitate nuclear transport. Tkachenko et al.43 combined

a receptor mediated endocytosis (RME) peptide and nucleus localization signal (NLS)

adapted from adenovirus into a long peptide and then loaded onto gold nanoparticles that

were already modified with a shell of bovine serum albumin (BSA). Such hybrid

nanoparticles were able to target the nucleus of human hepatocarcinoma cells HepG2.

Moreover, it was shown that BSA-gold nanoparticles conjugated with two individual

peptides have higher efficiency in targeting the nucleus than that with the longer combined

peptide. The Arg-Gly-Asp (RGD) motif, naturally found in a number of proteins including

laminin and fibronectin, has been used to specifically target several cell lines. RGD interacts

with several surface receptors, most prominently the CD51 family. HeLa cells strongly

express the CD51 receptor at a level of ~ 200,000 antibody binding capacity per cell44. The

most commonly used NLS peptide, the SV40 large T-antigen-derived NLS (PKKKRKV) can

facilitate nuclear uptake of nonnuclear proteins with a molecular weight up to 465 kDa45.

13
Ludtke et al used NLS-streptavidin to interact with biotinylated DNA. Addition of the NLS

peptide increased both nuclear uptake and expression of DNA after microinjection into the

cytosol of HeLa cells46. The 21-residue peptide carrier Pep-1, consisting of the hydrophobic

tryptophan-rich domain for efficient cell membrane translocation and the hydrophilic lysine-

rich domain derives from the SV-40-T antigen nuclear localization sequence to facilitate

solubility and nuclear targeting. The Pep-1 has been shown to be extremely efficient in the

targeting of proteins into cells independent of endocytosis47.

There are also other types of peptides that have been extensively studied. Among

them are the cell penetrating peptides (CPP), also known as protein transduction domain

(PTD). CPPs are typically used to refer to a class of small (<20 amino acid) cationic peptides

that can traverse the plasma membrane of many mammalian cells. The importance of these

peptides lies in their ability to uniformly transport large, biologically active molecules, such

as proteins or oligonucleotides, into mammalian cells.

The three most widely studied CPPs are from the Drosophila homeotic transcription

protein antennapedia (Antp, RQIKIWFQNRRMKWKK), the herpes simplex virus structural

protein VP22, and the human immunodeficiency virus 1 (HIV-1) transcriptional activator

TAT (YGRKKRRQRRR) protein 48. The third helix of the Antennapedia homeodomain has

been shown to form stable non-covalent complexes with small oligonucleotides and to

facilitate their internalization in a non-endosomal fashion. The NLS domain of TAT has been

shown to promote nuclear targeting of proteins and DNA. TAT peptide covalently attached

to liposomes promotes rapid delivery of DNA, independent of the endosomal pathway. In

contrast, oligomers of the arginine-rich motif of HIV-1 TAT protein have been reported to

14
form stable particles with DNA through non- covalent interactions, but promote their

delivery into cells through the endosomal pathway.

The mechanism by which peptides cross the plasma membrane and enter the cells

remains an active area of investigation. The fact that neither sequence inversion nor synthesis

with D-amino acids ablates the function of TAT and Antp indicates that they do not enter the

cell by interaction with a chiral receptor49. Instead, it seems likely that an electrostatic

interaction takes place between cationic PTDs and the negatively charged plasma membrane.

Early reports provided evidence that the uptake of PTDs, such as TAT and Antp, occurs in an

endocytosis-independent fashion. However, recent work has indicated that some studies on

the visualization of TAT uptake by standard methodologies, particularly methanol fixation,

are particularly prone to artifacts.

1.4.3 Therapeutics: antisense oligonucleotides

In this research, we have focused on using antisense oligonucleotides modified by

nanoparticle vectors to alter RNA splicing7. Antisense oligonucleotides have attracted

attention as promising therapeutic agents to treat cancer10, 50, 51. Gene therapy and delivery of

oligonucleotides as drugs that will modify protein expression present a new approach to the

treatment of cancer. Antisense oligonucleotides are short sequences of nucleotides (DNA,

RNA or a chemical anologue) typically with 20 or fewer bases, which are devised to be

complementary to a specific type of mRNA or its precursors (pre-mRNAs).

Alternative RNA splicing is in large part responsible for the diversity of gene

products in human and mammalian cells. The ability to manipulate alternative splicing using

antisense nucleotides makes it an important approach to altering gene expression52. In

15
diseases resulting in a loss of gene function even a modest increase in correct splicing and

gene expression may have significant therapeutic value. This concept has been successfully

demonstrated by using modified oligonucleotides to alter RNA splicing in the β-thalessemia

gene7. The delivery of a therapeutic oligonucleotide to a target in the RNA spliceosome can

have a profound impact on the survival of a cancer cell.

There are a number of obstacles that have to be solved when introducing

oligonucleotides into cells. First of all, oligonucleotides will be degraded by nucleases both

in the cytoplasm and in the nucleus. The lifetime of oligonucleotides can be enhanced by

chemical modification of the phosphodiester backbone and (deoxy)ribose sugars. Modified

oligonucleotides such as 2’-O-methylphosphonate, phosphorothioate, morpholino and

peptide nucleic acid (PNA) (Figure 1.4) have shown strong resistance against nuclease

degradation50, 52. Also, polymers such as PEG can be used to protect oligonucleotides.

Another limitation for oligonucleotides as antisense therapeutics lies in the low ability

of targeting cell, especially in the cases where nuclear targeting is required. By incorporation

of targeting peptides and antisense oligonucleotides onto one single nanoparticle, antisense

oligonucleotides should be able to traverse multiple cellular membrane and reach the nucleus

of the cells.

16
Figure 1.4 Chemical structure of naturally occurring and modified oligonucleotides.

1.5 Strategies to prepare nanoparticle-based delivery vectors conjugates.

In order to attach cell-targeting peptides and therapeutics onto nanoparticle surfaces,

effective methods for functionalizing nanoparticles have to be established. Two strategies

will be discussed below. One involves the covalent coupling of cysteine-terminated peptides

directly to a particle surface via a sulfur-gold bond (Figure 1.5). This approach takes

advantage of the ease with which the citrate layer on gold nanoparticles can be displaced by a

thiol on the gold surface53 and it affords a simple procedure that produces particles with a

17
high surface coverage of biomolecules. However, many peptides are sparingly soluble in

aqueous solution or cause aggregation when attached to gold nanoparticles.

Our solution to solve this problem is the use of stabilizing molecules like poly

ethylene glycol (PEG). PEG is one of the most popular polymers among steric stabilizers and

has been widely investigated for the covalent modification of biological macromolecules and

surfaces for many pharmaceutical and biotechnical applications54-57. PEG has the remarkable
58, 59
ability to stabilize gold nanoparticles in high ionic strength solutions . PEG can be

obtained with low polydispersity, and can easily be activated for conjugation. Citrate-

stabilized gold nanoparticles are mixed with PEG-thiols, thiol-terminated oligonucleotides

and cysteine-terminated peptides. The moiety of PEG-thiols prevents the aggregation of the

particles. An alternative route for this approach is the sequential addition of peptides and

PEG molecules. One can adjust the pH of the colloids to ~ 10 to avoid aggregation of the

particles before the addition of peptides. When the chemical equilibrium is reached between

the adsorbed thiols and free thiols, the PEG-thiol can then be added 30, 60. The PEG-thiol can

then block the remaining sites on the gold surface while also stabilize the pegylated gold

nanoparticles in high ionic strength solutions. The other way is to use PEG to stabilize the

nanoparticle followed by the addition of peptides. This strategy will be studied in chapter 3.

Figure 1.5 Covalent coupling of PEG and peptides to a nanoparticle surface.

18
 The second strategy involves the use of proteins. In our previous research43, 61
,

peptides have been coupled to bovine serum albumin (BSA) via the bifunctional crosslinker,

3-maleimido benzoic acid N-hydroxysuccinimide (MBS) (Figure 1.6). Attaching peptides to

BSA prior to immobilization on gold nanoparticles creates extra chemical reaction and

purification steps, but can increase peptide solubility and sol stability. However, such BSA-

coated nanoparticles easily aggregate upon the addition of certain basic peptides. This

observation has forced us to find alternative nanoparticle modification strategy.

Figure 1.6 The synthesis of peptide-modified gold nanoparticle conjugates for cellular
targeting. A surface lysine residue of BSA is labeled using MBS.

Streptavidin (SA) is a tetrameric protein from Streptomyces avidinii that has a strong
–15
biological and noncovalent affinity with the vitamin biotin (Kd=10 M)62. SA has been

widely used in biological systems and can be easily attached to colloidal gold. SA-conjugated

gold nanoparticles of different sizes are readily available. Figure 1.7 illustrates the synthetic

route to prepare nanoparticle vectors by making use of the SA-biotin interaction.

19
Figure 1.7 The synthesis of (peptide+oligonucleotide)-modified gold nanoparticle conjugates
via streptavidin-biotin interaction.

1.6 Size selection

The choice of the size of gold nanoparticles is of importance. Depending on the

targeting site and the intracellular uptake pathway, the size limitations for delivery vectors

vary. Endocytosis is achieved only if the delivery vector is 200 nm or less in diameter. If

transport of molecules from the cytoplasm to the nucleus occurs through the nuclear pore

complexes (NPCs), the upper limit in particle diameter for transport through the NPCs is

about 39 nm. Most delivery vectors are either quite large (50-1000 nm), limiting nuclear

delivery to nondividing cells, or are extremely small (e.g. dendrimers), limiting the number

of functions that can be incorporated. Those considerations suggest that a particle with the

diameter in the range of 10 – 30 nm is desirable as a nuclear delivery vector in our research.

1.7 Intracellular transport and processing of synthetic oligonucleotide vectors

DNA is generally maintained in the nuclei of cells, which do not have a mechanism

for importing DNA from their surroundings. Efforts to do so artificially must overcome some

cellular barriers. Thus, a detailed understanding of the uptake pathways and subsequent

20
intracellular trafficking of nonviral vectors is necessary for improving the design of

nanoparticle-based synthetic vectors.

1.7.1 Cellular binding and internalization

The vectors should be able to bind to cells to allow cellular uptake. If the vectors have

a net positive surface charge, the charge readily induces adsorption onto negatively charged

cell membranes. However, this form of binding is indiscriminate and does not allow targeted

delivery to target cells (for example, tumor cells). If targeting to a particular cell is desired, it

will be necessary to reduce the background uptake due to non-specific binding. This implies

the use of negatively charged or neutral particles, so that uptake by binding of polycations to

the cell membrane is minimized.

Endocytosis or uptake is characterized by the internalization of molecules from the

cell surface into internal membrane compartments. Most nonviral vectors exploit the

endocytic pathways for uptake and subsequent processing within the cells. There are four

types of pathways of endocytosis that have been described based on their morphological

characteristics including clathrin-mediated endocytosis, macropinocytosis, non-coated

endocytosis and caveolae.

Clathrin-mediated endocytosis is the major and best characterized pathway for uptake

of receptor-ligand complexes (A)63. The main defining feature of this well-known pathway is

the recruitment of soluble clathrin from the cytoplasm to the plasma membrane to form

clathrin-coated vesicles. Clathrin-coated vesicles are uncoated after endocytosis and then

fuse with the early endosomes64. The most common examples of molecules that are

21
internalized by this pathway are cholesterol-laden low-density lipoproteins (LDL) that bind

to LDL receptors and the iron-bearing transferring (Tf) that binds to Tf receptors65, 66.

Macropinocytosis refers to the formation of large and heterogeneous endocytic

vesicles, ’macropinosomes’ of irregular size and shape, generated by actin-driven

envagination of the plasma membrane67. The mechanism may be responsible for uptake of

large lipoplexes or polyplexes by dividing cells in culture. It has been demonstrated that the

uptake of the TAT peptide and its cargo occurs by macropinocytosis68.

Non-coated vesicle formation can contribute to bulk pinocytosis and some receptor-

mediated events. Although most receptors are clearly internalized by clathrin-coated pits,

other pinocytic pathways are capable of selective receptor meditated endocytosis events and

their use is a promising approach for introduction of macromolecules into defined cell

populations. For example, hepatocytes exclusively express large numbers of high affinity cell

surface receptors that bind to and subsequently internalize asialoglycoprotein69.

Caveolae are small lipid-enriched domains of the plasma membrane, which can be

identified by the presence of the marker protein caveolin. Caveolae have been implicated

particularly in endocytosis and transcytosis involving uptake into endothelia from the

vasculature. Some receptor mediated endocytosis takes place preferentially into caveolae

including folate uptake and some 7-transmembrane domain cell surface receptors. In general,

caveolae are highly stable and are slowly internalized. Caveolae uptake is a nonacidic and

nondigestive route of internalization. They do not suffer pH drop and can be directly

transported into the Golgi or endoplasmic reticulum, thus avoiding normal lysomal

degradation67.

22
1.7.2 Escape from the degradative pathway

For the clathrin-coated pit pathway, several intracellular barriers need to be crossed

inside the cells before the foreign DNA can be transcribed and translated. One intracellular

barrier encountered is the endosomal compartment. The entrapment of internalized vectors

in endocytic compartments prevents further cellular transport toward the nucleus, and will

often result in degradation of DNA in the endosomal/lysosomal compartments. Therefore,

the delivery vector should manage to escape the endosomal compartment in order to reach

the cytosol and, from there, the nucleus. Viruses have developed sophisticated protein

machinery to cross the endosomal membranes. After formation, the endosomes begin to

reduce their internal pH to ~ 5.5 due to the activation of proton pumps. The membranes of

the endosomes should be destabilized to allow translocation of the carriers into the cytosol.

This has become a very important strategy in the design of synthetic delivery vectors. For

example, synthetic peptides that mimick the amino-terminal fusion portion of the influenza

virus hemaglutinin have been used to facilitate endosomal escape. Cationic polymers with

high buffering capacity, such as poly(L-lysine) and poly(ethylenimine) (PEI) are thought to

escape endosomes according to the “proton sponge hypothesis”, where the buffering pH of

the endosomes promotes massive proton accumulation and the passive entry of chloride

ions and water. The resulting rapid osmotic swelling and endosomal membrane rupture

allows endosomes escape of delivered nucleic acid cargo molecules70.

1.7.3 Cytosolic trafficking

Those nucleic acid carriers that manage to escape the endosomal compartments are

then challenged by the complex environment of the cytoplasm. Direct microinjection of

23
DNA into the cytoplasm indicated that the half-life of DNA in the cytoplasm is ~ 90 min20.

The cytoplasm is composed of a fluid portion (the cytosol) in which a mesh-like network of

microfilaments and microtubules (the cytoskeleton) and several different subcellular

organelles are embedded. The diffusion of macromolecular or particulate systems within this

medium may be very slow. The cell uses molecular motors to transport vesicles, organelles

and other colloidal structures71. Dyneins, for instance, move vesicles in the inward direction,

normally associated with the endocytic pathway to facilitate transport towards the nuclear

envelope. Such active transport will greatly reduce the cytosolic residence time and enhance

survival of the DNA inside the harsh environment of the cytoplasm, which harbors several

nucleases.

1.7.4 Nuclear delivery

The nuclear envelope is a double-membrane enclosure that prevents the free passage

of large molecules from and into the nucleus. In general, there are two paths to nuclear entry-

one through the NPC and the other via nuclear disassembly that occurs during cell division.

During mitosis, the nuclear barrier breaks down and thus levels of transfection were

increased in dividing cells. Most cells do not divide or divide only slowly. Passive diffusion

through NPC is determined mainly by the size of the molecules, with rates of diffusion

inversely related to the mass. Small molecules (<40 kDa) can diffuse freely through the pores

of the nuclear pore complexes (NPC), whereas larger molecules and particles (up to 40 nm in

size) can only be imported through the NPC by an active transport mechanism. An active

transport mechanism is needed to carry the DNA from the cytosol into the nucleus.

24
 The nuclear import machinery consists of cytoplasmic karyopherins that recognize

and bind specific amino-acid sequences in karyophilic proteins called nuclear localization

sequences (NLS). They then dock onto the cytoplasmic side of the NPC, and the complex is

then translocated into the nucleus in an energy-dependent way. The nuclear import of DNA

can be enhanced by using synthetic NLS peptides. Conventional methods of conjugating the

NLS to DNA include electrostatic binding of cationic NLS with DNA or covalent

conjugation. Different strategies yield different outcomes in terms of transfection efficiency.

The most commonly used NLS peptide is the SV40 large T-antigen-derived NLS

(PKKKRKV). This cationic peptide binds DNA via electrostatic interactions. It can facilitate

nuclear uptake of nonnuclear proteins with a molecular weight up to 465 kDa45. Collas and

Alestrom associated SV40-derived NLS peptides (CGGPKKKRKVG-NH2) via ionic

interactions to luciferase-encoding plasmid DNA and microinjected the DNAYpeptide

complex into the cytosol of zebrafish embryos72, 73. Ludtke et al used NLS-streptavidin to

interact with biotinylated DNA. Addition of the NLS peptide increased both nuclear uptake

and expression of DNA after microinjection into the cytosol of HeLa cells46.

1.8 The splicing assay as a positive readout for nuclear targeting.

A simple, rapid and reliable method has to be used to evaluate the delivery of

antisense oligonucleotides into the nucleus of the cells. In our study, we have made use of

one type of model system developed by R. Kole to test oligonucleotide delivery7. Successful

modification of splicing by an antisense oligonucleotide indicates that the latter crossed the

membrane, entered the nucleus, found the target sequence in the newly transcribed pre-

mRNA, hybridized to it in vivo strongly enough to displace the splicing factors designed to

25
interact with the target sequence. Moreover, a new product, alternatively spliced mRNA is

generated, which can be easily detected on low background. Thus, apart from potential

clinical applications, modification of splicing can be used as a positive readout assay to

determine if functional oligonucleotides have been delivered to the splicing machinery within

the cell nucleus.

In this model system, HeLa cells are stably transfected with a recombinant plasmid

(pLuc/705) carrying the luciferase gene interrupted by a mutated human-beta globin intron 2

(IVS2-705). The mutation in the intron causes aberrant splicing of luciferase pre-mRNA,

preventing translation of luciferase, unless corrected by the presence of an antisense

oligonucleotide targeted to the splice site. The splice correction assay therefore measures the

ability of antisense oligonucleotides to correct splicing of an aberrant intron inserted into the

Luciferase gene.

My focus will be delivering the antisense oligonucleotide-modified nanoparticles into

the pLuc HeLa cell line and test the bioactivity of these particles. If the particles reach the

nucleus of the cells, the antisense, ON705 will correct the aberrant spicing of luciferase

caused by the mutation in pre-mRNA, thus, proper luciferase genes will be produced and the

correct luciferase protein will be expressed (Figure 1.8)

26
Figure 1.8 The splicing assay as a positive readout for nuclear targeting.

1.9 Summary

It is clear that the hybrid nanoparticle-based delivery vectors that we proposed have

advantages over viral vectors and non-viral delivery vectors. The choices of the carrier, cell-

targeting peptides, therapeutics and the bioactivity assay have been made and those particle-

based vectors have the potential to overcome the cellular barriers, target the nucleus of the

cells and thus correct the aberrant splicing of pre-mRNA, producing correct protein.

In the next chapter, gold nanoparticles, as the carrier for both the cell-targeting

peptides and therapeutics, will be discussed in the aspects of synthesis, functionalization,

optical properties and biological applications.

27
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33
Chapter 2 Gold Nanoparticles: Synthesis, Functionalization,

Stability and Biological Applications

34
 This chapter will review the synthesis and modification of gold nanoparticles of gold

nanoparticles. Stability of gold nanoparticles is one of the most important issues for

biological applications. Therefore, the fundamental understanding of the colloidal stability

and effects of polymers on the stability will be discussed in detail. Properties of gold

nanoparticles and their biological applications will be described at the end of this chapter.

2.1 A brief historic introduction

Colloidal gold is one of the most ancient subjects of scientific investigation. In

antiquity, colloidal gold was used for both aesthetic and curative purposes. Gold was used to

make ruby glass and for coloring ceramics since Glauber’s discovery in the mid 17th century

of “Purple of Cassius” and similar applications have continued up to the present1. Around

1600, Paracelus described a preparation of “aurum potable, oleum auri; Quinta essentia auri”

by the reduction of auric chloride (AuCl3) with an alcoholic extract of plants2. This red

mixture was an example of a gold colloid formed by condensation. It was one of the first

synthetic drugs and was called “Jin Tu” in Chinese and “Makaradhwaja” in Indian3.

However, by the eighteenth century, it was realized that the potable gold tinctures were

nothing but extremely finely divided gold floating in an oily fluid that was devoid of any

medicinal, therapeutic or economic value4. The major use of gold colloids in medicine in the

Middle Ages was to diagnose syphilis and it was not completely reliable5.

The English scientist Faraday (1791-1867) reported the formation of deep-red

solutions of colloidal gold by reduction of an aqueous solution of chloroaurate (AuCl4-) in

1857 using phosphorus in CS2 (a two-phase system)6. He discovered the effect of electrolytes

on colloidal gold and the protective effect of gelatin and other macromolecules. Some of his

35
original gold preparations are still preserved at the Royal Institute in London. The term

“colloid” (derived from the Greek word for glue kolla4, which became the French word

colle1) was coined shortly thereafter by Thomas Graham (1805-1869) in 1861 to distinguish

those materials in aqueous solution that would not pass through a parchment membrane from

those that would7. Gold had been known for its healing capabilities for the heart and

improved blood circulation since 1885. And gold has been used to treat arthritis continuously

since 1927

Colloids have received wide attention because of the importance of colloids to

industrial processes and biochemistry. In the past decade, gold colloids have been the subject

of a considerably increased number of books and reviews in the context of emerging

nanoscience and nanotechnology. Gold nanoparticles are expected to be key materials and

building blocks as well as playing a great role in medicine in the near future1, 8-12.

2.2 Chemical synthesis of colloidal gold

Since the pioneering work of Faraday on the synthesis of stable aqueous dispersions

of gold nanoparticles, there have been a large number of reports on the synthesis and

assembly of gold nanoparticles both in aqueous and organic solutions. In all the methods

developed to date, the emphasis had been to synthesize gold nanoparticles in a simple way

with uniform and controllable size. All of these methods use tetrachloroauric acid (HAuCl4)

but differ considerably with reducing agents, order of reagent addition, physical parameters

such as concentration, temperature, and mixing rate and the resulting size of the

nanoparticles. The final size of the gold nanoparticle is determined by the number of

icosahedral nuclei formed at the beginning of the reaction compared with the subsequent rate

36
of shell condensation. The diameter of the particle can be decreased by increasing the amount

of reductant used, decreasing the volume, increasing reaction temperature and increasing

agitation to change the degree of reactant mixing during the nucleation step.

2.2.1 Citrate reduction in aqueous solutions

The method of citrate reduction to synthesize gold nanoparticles in water was

pioneered by Turkevich et al. in 195113, and modified by Frens in 197314. Citrate reduction

has been the most popular method and is still widely used today. The typical procedure is to

boil under refluxing conditions an aqueous solution of HAuCl4. While stirring vigorously,

sodium citrate is quickly added, resulting in color changes from faintly yellowish to clear,

grey, purple and dark purple.

The citrate first acts as a reducing agent. It reduces Au3+ ions to zero-valent gold

atoms. As more and more of these gold atoms form, the reaction solution becomes

supersaturated, and gold gradually starts to precipitate in the form of sub-nanometer particles.

The rest of the gold atoms that form stick to the existing particles, and, if the solution is

stirred vigorously enough, the particles will be fairly uniform in size. The negative citrate

ions later are adsorbed onto the gold nanoparticles. The adsorption of these molecules to

nucleated particles lowers the free energy of the surface and therefore, the reactivity of the

particles. In the meantime, the citrate ions introduce surface charge onto the particles, which

repels the particles from contacting each other and thus prevents the aggregation of the

particles. Varying the ratio of citrate to HAuCl4 can control the size and the distribution of

gold nanoparticles. Higher ratios yield smaller particles.

37
2.2.2 Reduction from non-aqueous solutions

Brust et al. reported the facile synthesis of alkanethiol-stabilized colloidal gold

nanoparticles that are stable in organic solvents in 199415. Starting from an aqueous solution

of AuCl4-, the tetrachloroaurate ions were transferred to toluene using tetraoctylammonium

bromide (TOAB) as the phase-transfer catalyst and reduced with an aqueous solution of

sodium borohydride (NaBH4) in the presence of dodecanethiol (C12H25SH). 1-3 nm diameter

colloidal gold coated with thiols was formed in the organic phase and subsequently isolated

by vacuum evaporation or by precipitation with methanol. The overall reaction was

summarized by eqns. (1) and (2), where source of electrons is BH4-

AuCl4- (aq) + N(C8H17)4+ (C6H5Me) N(C8H17)4+ AuCl4-(C6H5Me) (1)

AuCl4-(C6H5Me) + nC12H25SH(C6H5Me) + 3me-

4mCl-(aq) + [Aum(C12H25SH)n] (C6H5Me) (2)

The conditions of the reaction determine the ratio of thiol to gold, n/m.

Brust et al. extended this method to p-mercaptophenol-stabilized gold nanoparticle

into a single phase system16. Subsequently, synthesis of thiol-based stabilization of gold

nanoparticles, also called monolayer-protected clusters (MPC) became popular. This method

has had a considerable impact in this field. Later, numerous new thiol-, amine-, silane-,

phosphine-, and disulfide-based capping ligands have been identified, and several techniques

have emerged for the exchange of capping ligands. This effectively allows the functionality

and chemical properties of the ligand shells to be tuned.

Chen et al.17 reported that gold nanoparticles reduced by borohydride and capped

with mercaptosuccinic acid (MSA), exhibit the additional benefit of being dispersable in

38
water. The sizes of the nanoparticles were controllable within the 1-3 nm range by varying

the mol ratio of MSA to gold.

2.3 Strategies for functionalization of gold nanoparticles

Functionalization of gold nanoparticles is a prerequisite to their use as multifunctional

reagents, chemical sensors and therapeutic delivery vectors. There are four common

strategies for tailoring the composition of the monolayer on nanoparticles and the functional

groups exposed at the monolayer-solvent interface. They are (1) forming the nanoparticles

directly in the presence of ω-functionalized thiols, (2) exchanging an existing ligand for a ω-

functionalized thiol, (3) modifying the original thiol covalently by an interfacial reaction, and

(4) coupling biomolecules and nanoparticles through electrostatic interactions. Each of these

approaches will be addressed in the following sections.

2.3.1. Formation of nanoparticles in the presence of thiols

The original Brust-Schiffrin method opened an avenue to the synthesis of gold

nanoparticles with a variety of functional thiol ligands because this method tolerates

considerable modification of the protecting ligand structure. Some alkanethiols and other

organic soluble thiols can survive the reductive conditions used to prepare nanoparticles and,

therefore, can be used to protect the nanoparticles during synthesis (Figure 2.1).

39
Figure 2.1 Formation of gold nanoparticles by reduction of HAuCl4 in the presence of thiols.

Since water-soluble nanoparticles are desirable for biological applications, many

preparations have been developed that use thiols with hydrophilic, polar headgroups17-20. For

example, mercaptosuccinic acid (MSA) can serve as a stabilizer during borohydride

reduction of HAuCl4 to give 1-3 nm, water-dispersible gold nanoparticles that are stabilized

by the charge-charge repulsion of the carboxylate ions17. Glutathione18, tiopronin(N-2-

mercaptopropionyl-glycine)19, coenzyme A (CoA)19, trimethyl (mercaptoundecyl)

20
ammonium, and thiolated derivatives of PEG have all been used as thiol-based water-

soluble stabilizers during the formation of gold nanoparticles with a variety of reductants.

Arenethiolate16, 17, 21, 22 and (γ-mercaptopropyl)trimethyloxysilane (MPS) ligands23 have also

been used to prepare small gold nanoparticles. Sterically bulky ligands (larger footprint) tend

to produce smaller gold core sizes, suggesting a possible steric connection to the dynamics of

core passivation.

40
2.3.2. Ligand exchange methods

Figure 2.2 General scheme for the ligand-exchange reaction between alkanethiol-gold
nanoparticles of the Brust type and various functionalized thiols.

Displacement of one ligand for another is a second strategy for modifying the organic

surface of nanoparticles after their formation (Figure 2.2). Ligand place-exchange methods

are particularly useful if the desired ligand is not compatible with the highly reductive

environment required for forming nanoparticles or if the desired ligand is particularly

valuable (or simply not commercially available) and cannot be used in the excess necessary

for stabilization during synthesis.

Generally, gold (Au) nanoparticles with alkanethiolate monolayers (RS) can be

functionalized with XR’S groups by the reaction

n(XR’SH) + (RS)m Au n(RSH) + (XR’S)n(RS)m-n Au

where n and m are the numbers of new and original ligands, respectively. This strategy

produces gold nanoparticles stabilized by mixed monolayers of unsubstituted and ω-

substituted alkanethiolates (X = Br, CN, Vinyl, Ferrocene, Ph, OH, CO2H, CO2CH3,

Anthraquinone)24, 25. The rate and equilibrium stoichiometry (n) of the reaction are controlled

by factors that include their relative steric bulk of the ω-functional group, the chain lengths

and the concentrations of the initial protecting (RS) and incoming (R’S) thiolates25, 26. The

41
rate of the reaction decreases with an increase in the size of the entering ligand and the chain

length of the protecting monolayer. Ligand exchange is an associative reaction and yields the

displaced ligand in solution as a thiol. Disulfides and oxidized sulfur species are not involved

in the reaction.

Simple thiols can be exchanged for more complex thiols or disulfides. Ligand

exchange can be accomplished via either simultaneous or stepwise exchange25. There are

also several recent reports of solid-phase place-exchange reactions with thiol ligands

displayed on Wang resin beads27, 28.

Thiols can displace other ligands weakly bound to gold (e.g., phosphines29 and citrate

ions). This is especially useful when functionalizing large gold nanoparticles (>5 nm) cannot

be formed directly with thiols29-32. For example, dodecanethiol has been used to extract gold

nanoparticles from water into organic solvents33, 34. Caruso and co-workers demonstrated the

synthesis of water-soluble nanoparticles modified with 11-mercaptoundecanoic acid (MUA),

for example, through ligand exchange from hydrophobic n-alkanethiols in toluene35. Chen et

al30 showed two-step functionalization of neutral and positively charged thiols onto citrate-

stabilized gold nanoparticles. The citrate is first displaced by thioctic acid (TA), followed by

an exchange with thiols. The resulting nanoparticles are successfully functionalized by

negatively charged (carboxylate), neutral (crown and cyclodextrin), and positively charged

(pyridinium and ammonium) moieties. More recently, Amiji et al36 demonstrated the use of

hetero-bifunctional poly(ethylene glycol) spacer (PEG, MW 1500) for functionalizing citrate

coated gold nanoparticles for cellular delivery (Figure 2.3).

42
Figure 2.3 Surface functionalization of gold nanoparticles with coumarin-PEG-thiol through
ligand exchange.

2.3.3. Covalent modification

Terminal functional groups of the ligand on nanoparticles can undergo many

synthetic transformations. There are several routes for covalent coupling on nanoparticle

surfaces: straightforward reactions with a functional terminus; activation of surfaces for

reactions; reactions that break covalent bonds and surface-initiated polymerization11. Many

reactions have been used to functionalize nanoparticles including amide and ester coupling

reactions37, cross-metathesis (forming carbon-carbon bond under mild reaction conditions)38,

peptide coupling reactions28, 39, and nucleophilic substitution reactions40.

In an early example16, 41, Schiffrin reported the esterification of p-mercaptophenol-

protected MPCs using propionic anhydride. Murray and co-workers demonstrated the use of

chemical reactions to modify the terminal functional groups of alkanethiolates on

nanoparticles and examined the steric effects with SN2 reations37. Mrksich et al.42 have

shown that SAMs presenting maleimide functional groups react in good yield with

biologically active ligands (peptides and carbohydrates) having thiols.

Polymer coatings can contribute durability and toughness to monolayers on

nanoparticles. Coupling silane-functionalized nanoparticles with mercaptosilane monomers,

forming cage-like structures surrounding the gold core was demonstrated23. Mirkin’s group

43
has employed ω-norbornenyl-functionalized particles to grow polymeric shells using a

ruthenium carbene ring opening metathesis polymerization (ROMP) catalyst; these shells can

be functionalized with virtually any norbornenyl-containing monomer43.

2.3.4 Electrostatic binding

In the case of nanoparticles stabilized by anionic ligands such as citrate or lipoic acid,

the biomolecules are often coupled to gold nanoparticles through noncovalent electrostatic

interactions. For instance, citrate stabilized gold nanoparticles have been functionalized with

IgG44, BSA45, 46 and streptavidin44, 47, 48 at high pH values (above the isoelectric point of

citrate ligand), which allows an effective binding between the positively charged amino acid

side chains of the protein and the negatively charged citrate groups of the gold nanoparticles.

Furthermore, certain proteins can be used as a hetero bifunctional linker. For

example, the Franzen and Feldheim groups46 have used BSA as a bridge to crosslink various

peptides to citrate-coated gold nanoparticles. Those particles traverse cell membranes and

target the nucleus of certain cell lines. Streptavidin (SA) is also used commonly to couple

gold nanoparticles with biotin-DNA47 and other biomolecules44, 49

because of the high

binding affinity of the SA for biotin 49. These have been discussed in chapter 1.

2.4 Stability of colloidal gold

Colloidally stable means that the particles do not aggregate at a significant rate. An

aggregate is used to describe the structure formed by the cohesion of colloidal particles.

Aggregation is the process or the result of the formation of aggregates. When a sol is

colloidally unstable, the formation of aggregates is called coagulation or sometimes

44
flocculation. These terms are often used interchangeably, but we will reserve the term

flocculation to refer to aggregation induced by a polymer. More specifically, flocculation is

used for the formation of rather loose aggregates of particles linked together by a polymer as

distinct from coagulation in which the particles come into lose contact as a result of changes

in the electrical double layer around the particles50.

2.4.1 Critical Coagulation Concentration (CCC)

Gold colloids are usually sensitive to electrolytes (salts). Addition of an electrolyte

can cause colloids to aggregate. A defined concentration of salt to induce coagulation is

called Critical Coagulation Concentration (CCC). To determine the CCC of gold colloids, a

series of tubes containing identical concentrations of gold nanoparticles are prepared.

Increasing volumes of a salt stock solution are added to each sample with the final salt

concentrations ranging from 20 mM to 1 M. Following mixing, the tubes are then allowed to

incubate for equilibration for a few hours. Aggregation is assessed by monitoring changes in

the characteristic gold nanoparticle plasmon frequency by UV-visible absorption

spectroscopy. There is also clear evidence of coagulation, such as the settling out of the

dispersed phase, or a color change due to a broadening of the plasmon band. The threshold

salt concentration in the gold sol, which causes the rapid aggregation of the particles, is

determined as the CCC.

There are several factors that influence the CCC: (1) the equilibration time allowed to

elapse before the CCC is determined, (2) the uniformity or the polydispersity of the particles,

(3) the surface potential of the particles, (4) the Hamaker constant (the characteristic

parameter for materials) of the particles, and (5) the valence of the ions. In CCC assay, (1)

45
through (4) remain constant, so the CCC is a quantitative measure of the effects of the added

ions.

2.4.2 Van der Waals forces

One of the most important forces in colloid chemistry, namely, van der Waals forces

will have to be addressed before we start to discuss stabilization mechanisms in this chapter.

The forces operate between macroscopic objects as well as between atoms and molecules.

These forces consist of three major categories known as Keesom interactions (permanent

dipole-permanent dipole interaction), Debye interactions (permanent dipole-induced dipole

interaction), and London interactions (induced dipole-induced dipole interaction). Spherical

colloids have negligible dipole moments. Therefore, the London force, or dispersion force is

the most significant of the three and is always present in the colloidal systems (like the

gravitational force). Hamaker theory, or pairwise summation of forces50 proposed an

approach to sum all interactions in the system. As a rough estimate, the Hamaker constant for

most material is in the range of 10-19 to 10-21 J50. Bargeman et al determined the Hamaker

constant of gold (4.53 x 10-19 J)51.

2.4.3 Electrostatic stabilization

Gold colloids are composed of an essentially internal crystalline core of pure gold,

which is surrounded by a surface layer of adsorbed ions (citrate, halides or carboxylates),

conferring the negative charge to the surface of gold. The surface charge together with the

counter ions, e.g. H+, K+, Na+, in the solution forms an electrical double layer. The electric

potential associated with the double layer decreases as a reciprocal function of distance

46
(proportional to 1/R) from the particle and approaches zero as the counterions gradually

reduce the effect of the field created by the surface. When two particles approach each other,

the overlap of the counterions in the diffuse layer gives rise to Coulombic repulsion force

between the particles and repulsion potential.

This was actually explained with DLVO theory, which was proposed independently

by Derjaguin and Landau (1941) of the former Soviet Union and Verwey and Overbeek

(1948) of the Netherlands. It accounts for the energy changes when two particles approach

each other and involves estimation of the van der Waals attraction and electrostatic repulsion

as a function of interparticle distance. These estimates are then added so as to yield the total

potential energy. Figure 2.5 A illustrates the potential energy between two particles as a

function of the distance between two particles. To be concise, if the Coulombic repulsion

force is high enough to counteract the van der walls force, then the electrostatic repulsion

will prevent particles from aggregation.

2.4.4 Steric stabilization

The stability of colloidal gold can often be improved with the use of macromolecules

such as polymers or oligomers, which is known as steric stabilization. Steric stabilization is

more widespread and has certainly been used for a great deal longer than electrostatic

stabilization. The early makers of inks and paints were well aware of the value of certain

natural gums in promoting the stability of the pigments and the early colloid scientists

referred to this phenomenon as protection4.

The free energy of mixing of the adsorbed molecular chains as the particles come

together can be expressed as ∆Gmix = ∆Hmix – T ∆Smix. A positive value of ∆Gmix (repulsion)

47
can arise from either a positive value of ∆Hmix (enthalpic stabilization) or a negative value of

∆Smix (entropic stabilization) or both. Enthalpically stabilized systems can be made to

coagulate at high temperatures (∆Smix >0) and the opposite is true of entropically stabilized

systems50. The adsorbed molecules are firmly attached to the surfaces of the particles so that

they can neither desorb nor migrate from the encounter region during a collision, which

causes a decrease in entropy and thus an increase in free energy (Figure 2.4). The second

effect is due to the local increase in concentration of adsorbed molecules as the two

protective layers begin to interpenetrate. This results in an osmotic repulsion as the solvent

will reestablish the equilibrium by diluting the macromolecules and thus separating the

particles.

Figure 2.4 Schematic representation of steric stabilization of colloid particles.

A typical potential energy diagram for the closest approach of two sterically

stabilized particles is displayed in Figure 2.5 B.

48
Figure 2.5 Plot of potential energy V(d) vs interparticle distance (d) for electrostatic
stabilization (A) steric stabilization (B).

2.4.5 Electrosteric stabilization

A more complicated effect can arise if a polyelectrolyte is used as a stabilizing ligand

- both electrostatic and steric forces contribute to colloidal stability. The polar head group on

polyelectrolytes produces an electric double layer; resulting electrostatic repulsion while a

lypophilic side chain provides steric repulsion. Adsorption of such ligands to particles will

result in a large particle net charge and thus an effective electrostatic stabilization mechanism

to add to the steric effect. This combination is known as electrosteric stabilization, a

phenomenon widely encountered in biological systems. The charge will be distributed along

the adsorbed polymer chains. At low bulk electrolyte concentrations, the double-layer

repulsion will be significant, and the chains will repel each other, causing polymer “hairs”

attached to the particle surface to stand on end. At high bulk electrolyte concentrations, the

49
hairs may collapse back towards the particle surface, thus diminishing the effective particle

diameter as the double-layer repulsion between particles is reduced.

2.4.6 Polymeric stabilization and flocculation

The addition of polymers to a colloidal suspension can cause a variety of effects.

Depending on the concentration of the input polymer, the size and the chemical nature of the

polymer, the added molecules can induce either flocculation or stabilization of the

suspension. The effects of polymer, free or attached, on the stability of colloidal dispersions

are presented schematically in Figure 2.7 and will be discussed in the following section.

Figure 2.6 Effects of polymer chains on colloidal dispersions.

2.4.6.1 Effects of adsorbed polymer

If the polymer adsorbs to the surface, two different types of effects can be identified:

bridging flocculation and steric stabilization.

50
 When one polymer molecule can simultaneously adsorb onto the surfaces of more

than one particle, the polymer will hold the particles together (Figure 2.7). Such bridging

links the particle together into loose aggregates, which often sediment rapidly or are easily

removed by filtration. This effect is called bridging flocculation52. This phenomenon is

usually the result of strong adsorption at very low polymer concentration (p.p.m.) using

polyelectrolytes of high molecular weight (say, a few million)53.

Figure 2.7 Bridging flocculation of colloidal particles by polymeric flocculants.

At higher polymer concentrations than the optimum for flocculation, the particles are

fully coated by polymer chains attached solely to one particle. If the polymers form a layer

around each particle, the polymer layers can give rise to a repulsive force, and steric

stabilization is then observed. Requirements for steric stabilization include that the adsorbed

and anchored polymer layer should be dense, thick, and completely cover the particles, in

order to prevent the particles from coming close enough that van der Waals forces will give

rise to a net attraction.

51
2.4.6.2 Effects of free polymer

Low concentrations of free polymer have no apparent effect on the stability of

colloidal particles. Free polymer effects occur only at moderate to high polymer

concentrations; increasing the free polymer concentration would result in the onset of

flocculation, which is named depletion flocculation54. An attractive force is created when the

particles approach so closely as to exclude polymer chains from the interparticle region so

that microreservoirs of essentially pure solvent are generated (Figure 2.8). Closer approach of

the particles is then favored because it leads to a reduction in free energy as the pure solvent

is pushed out from between the particles and mixes spontaneously with the bulk polymer

solution. Alternatively, one can view the depletion flocculation as an osmotic effect. The

bulk solution of the polymer exerts a compressive osmotic pressure on the particles. The

particles behave as a semi-permeable membrane and prevent the flow of the polymers into

the space between particles and so produces a compressive osmotic interaction.

Figure 2.8 Schematic representation of the origin of depletion flocculation.

52
 At higher polymer concentrations there would be a second effect arising from the

entropy penalty of compressing the free polymer chains between particles, if not all of the

chains are excluded from the gap. This would give a repulsive force and lead to depletion

stabilization. This effect can simply be understood in terms of the work required to create a

polymer free region between two approaching particles.

2.5 Properties and applications of gold nanoparticles

2.5.1 Physical-chemical properties and the use as a platform to display multiple

polyvalent ligands

Gold is a reasonably inert metal: it does not oxidize at temperatures below its melting

point; it does not react with atmospheric O2; it does not react with most chemicals. These

properties make it possible to handle and manipulate samples under atmospheric conditions.

On the other hand, gold nanoparticles can be easily synthesized by citrate reduction in an

aqueous solution or from an organic solution as discussed in the previous sections. Sizes of

the nanoparticles can be tuned reliably and routinely from 1 nm to 200 nm with <10 % size

dispersity55. They are also exceptionally easy to functionalize. They can be modified with

nearly any thiol-containing small molecule or with polymers (cysteine-teminated peptides,

ssDNA and ssRNA) via gold-thiol covalent bond. Gold binds thiols with a high affinity and it

does not undergo any unusual reactions with them56 and because of that, thiols also displace

adventitious materials from the surface readily.). Gold can be coated with macromolecules

such as proteins through electrostatic binding as well45. Gold can also be modified by

heterobifunctional crosslinkers, where one functional end can be utilized for attachment on

the gold nanoparticle surface while the other terminal functionality remains available for the

53
desired application. Gold nanoparticles have been modified with various ligands, including

carboxylic acids, phosphines, oligonucleotides, amines, proteins, enzymes and small drug

molecules57-62. Gold is biocompatible. Cells can function on gold surfaces without evidence

of toxicity46, 63. The future use of gold nanoparticles in clinical applications is envisioned64.

All of these properties make gold nanoparticles an excellent platform to display multiple

polyvalent ligands65.

Polyvalent interactions, the simultaneous binding of multiple ligands on one

biological entity (a molecule, a surface) to multiple receptors on another, occur broadly in

biological systems and are much stronger than corresponding monovalent interactions66.

Mimicking virus-like surfaces by employing gold nanoparticles as building blocks provides a

tool for biological studies. For example, Sengupta et al.67 demonstrated that a nanoparticle

formulation loaded with two different agents was more effective in suppressing tumor growth

than a single drug therapy. Moreover, combinations of two or more chemically distinct

ligands can be attached to one single nanoparticle to impart multiple functions often via

simple one-pot procedures. The combination of two short peptides (RME and NLS) on one

single gold nanoparticle has been shown to be more efficient for targeting the nucleus of

HepG2 cells than one long peptide with full sequence of RME and NLS45, 46.

2.5.2 Optical properties of gold nanoparticles and their biological applications

In metal nanoparticles, collective electron oscillations known as surface plasmons

(SPs) can be excited by light. The SPs show themselves as pronounced optical resonances in

the visible or UV parts of the spectrum. The resonance frequency of the oscillation, i.e., the

54
SP energy, is essentially determined by the dielectric properties of the metal and the

surrounding medium, and by the particle size and shape.

The red color characteristic of 10 –50 nm diameter gold nanoparticles arises from the

surface plasmon band (SPB), a broad absorption band in the visible region around 520~530

nm. Mie theory considers the collective dipole oscillations of the free electrons in the

conduction band contribute to the SPB of spherical particles68. The oscillations of the

electrons correspond to the electromagnetic field of the incoming light and occupy the energy

states immediately above the Fermi energy level (surface plasmon resonance). Only metals

with free electrons such as gold, silver and copper have SPB in the visible spectrum, which

produce intense color. The SPB maximum (λmax) and band width have been found to depend

on various characteristics of the nanoparticles, such as their size, shape and structure (spheres

or core/shell particles) in addition to the refractive index of the surrounding medium, which

is itself influenced by the presence of biomolecules at the surface of the particles. With an

increasing particle diameter, the peak shifts to a longer wavelength, while the width of the

absorption band relates to the size dispersity. When nanoparticles aggregate, the SPB

disappears as accompanied by the absence of the red color.

Gold nanoparticles have large extinction coefficients. For example, 20 nm diameter

has extinction coefficient of 108 M-1 cm-1 at about 520 nm. This is several orders of

magnitude larger than that of regular organic dyes. Moreover, gold nanoparticles have an

exceptional resistance to photodegradation.

All these optical properties make gold nanoparticles useful for many applications

including immunoblotting, biosensors, disease diagnosis and cell imaging4, 12, 69, 70.

55
 When gold nanoparticles come into close proximity, plasmon-plasmon interactions

cause dramatic changes in optical properties. Using appropriately conjugated nanoparticles,

this behavior can be exploited for DNA hybridization assays and immunoassays. For

example, Mirkin and co-workers71 reported the color change of oligonucleotide-modified 13

nm diameter gold nanoparticles from red to blue upon the addition of complementary

sequence oligonucleotide-modified nanoparticles, which pointed toward the use of gold

nanoparticles as DNA detection agents. Indeed, subsequent research has improved the

detection limit to as low as ~ 1 fM (typical fluorophore-based assay in picomolar range)72. A

similar approach has been used to develop a rapid immunoassay. Halas and co-workers73

uses gold nanoshells coated with antibodies to detect proteins in whole blood. These

nanoshells aggregate upon the addition of the target protein, accompanied by a corresponding

broadening of the extinction peak at 720 nm. This assay is simple, fast (10 min), and very

sensitive (88-0.88 ng/mL).

In addition, the use of gold nanoparticles as a Fluorescence Resonance Energy

Transfer (FRET) quencher is worth mentioning. One example involves a fluorophore (F)

labeled oligonucleotide in a bent conformation on a gold nanoparticle surface. In this

situation, the fluorophore is quenched by the nanoparticle. By addition of a perfectly

matching single-stranded DNA, restoration of the fluorescence occurs because of the more

rigid conformation of hybridized DNA (Figure 2.9)74.

56
Figure 2.9 Gold nanoparticle-based DNA biosensor as a molecular beacon.

Faulk & Taylor invented the immunogold staining procedure in 19714. Since then, the

labeling of targeting molecules, such as antibodies, with gold nanoparticles has

revolutionized the visualization of cellular components by electron microscopy. The light

extinction properties of gold nanoparticles (visible light extinction coefficients >108 M -1 cm -

1
at ~ 520 nm) has been exploited with various imaging techniques such as Differential
45, 46
Interference Contrast Light (DIC) microscopy or simple dark field microscopy75. Gold

nanoparticles (35 nm in diameter) modified the anti-EGFR antibody were incubated with

malignant cells and then found specifically and homogeneously bind to the surface of the

cancer cells with 600% greater affinity than to the noncancerous cells by simple dark field

microscopy75, 76. Gold nanoparticles modified with peptides have been used for investigating

nuclear transport trajectory in our research group45, 46. Those particles were examined by DIC

microscopy. Those studies help to understand the cellular process of gold nanoparticles.

2.6 Summary

Gold nanoparticles are one of the most studied inorganic particles. They are easily

synthesized in either an aqueous solution or organic phases with tunable sizes from 1 – 100

nm in diameter. Functionalization of gold nanoparticles is straightforward. Generally, gold

57
nanoparticles can be modified with various molecules from short peptides, oligonucleotides

to proteins and polymers. Unique physical-chemical properties and optical characteristics of

gold nanoparticles make possible wide applications in biomedicine and nanotechnology.

My research is to design, prepare, characterize and deliver multifunctional

nanoparticle vectors for cellular internalization and nuclear targeting. In the next chapter, we

try to understand how to prepare a stable and functional nanoparticle vector in cell media,

how to quantify the peptides and therapeutic loadings on one nanoparticle, how to resist

nonspecific binding of proteins to nanoparticles in a biological environment, and how to

quantify the number of nanoparticles internalized into cells.

58
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64
Chapter 3 Synthesis, Stability, and Cellular Internalization of

Gold Nanoparticles Containing Mixed Peptide-Poly(ethylene

glycol) Monolayers

65
3.1 Introduction

Nanometer-sized metallic, magnetic, and luminescent clusters have emerged as

promising materials for probing and manipulating cellular processes. Collectively, they offer

nanoscale platforms that can be functionalized with small molecule drugs, polymers, and

biomolecules, and they display a range of important physical properties such as high optical

extinctions (gold), stable photoemission (CdSe), superparamagnatism (CoFe2O4), and the

surface-enhanced Raman effect (silver). A few of the exciting applications of nanoclusters

documented recently are photothermal ablation of tumors with gold shells1, Her2 imaging

with quantum dots2-4, and magnetic resonance imaging of cells with iron oxide nanoparticles

conjugated to the HIV tat peptide5.

Applications of nanoclusters in vivo commonly employ antibodies or peptides for

selective cell targeting and internalization6, 7. For example, cell and nuclear targeting by 20

nm diameter gold nanoparticles conjugated to various peptide sequences has been

demonstrated8, 9. In earlier studies, peptides were first covalently bound to the protein bovine

serum albumin (BSA), which was subsequently adsorbed onto gold nanoparticles via

electrostatic interactions. The use of proteins such as BSA and streptavidin as linkers

between peptides or antibodies and nanoparticles is common. However, adsorption

thermodynamics and the stability of the resulting gold sol against aggregation in biological

media are highly dependent upon the particular peptide sequence chosen. The frequent

instability of peptide-BSA-gold nanoparticle conjugates in biological media prompted us to

explore PEG-based alternative formulation strategies for cell targeting nanoparticles. One

design strategy employs polyethyleneglycol (PEG) as a co-adsorbate for peptides attached to

66
gold nanoparticles. The synthesis, characterization, stability, and resistance to protein non-

specific binding of mixed peptide-PEG monolayers on gold nanoparticles are reported here.

3.2 Experimental Methods

3.2.1 Reagents and instrumental

All chemicals were used as received from the manufacturer without further

purification. Triethylamine and chloroform were purchased from Fisher Chemicals. Gold

nanoparticles (10, 20, 30, and 60 nm diameter) were purchased from Ted Pella. Fluorescein

isothiocyanate (FITC)-modified IgM and Rhodamine (Rh)-labeled BSA were purchased

from Fisher. All phosphate-buffered saline (PBS) solutions used were 10 mM unless noted.

All other solvents and chemicals were purchased from Sigma-Aldrich. All 1H NMR (400

MHz) spectra were recorded at 25.0 ºC on a Varian Mercury spectrometer. Infrared spectra

(KBr pellet) were taken using JASCO FT-IR-410 spectrophotometer. UV-visible absorption

spectra were acquired on a Hewlett-Packard 8453 Chemstation photodiode array

spectrophotometer with attached Chemstation software. Fluorescence intensity data were

obtained from a Perkin-Elmer luminescence spectrometer LS50B or BioTek FL-600 plate

reader. ζ-potential measurements were performed on a NanoZ Zetasizer with a 633 nm He-

Ne laser from Malvern Instrument UK, Inc. Dynamic light scattering (DLS) measurements

were obtained on a Malvern Zetasizer 1000. Inductively Coupled Plasma Optical Emission

Spectroscopy (ICP-OES) experiments were performed on a Perkin-Elmer Optima 2100DV

Optical Emission Spectrometer.

67
 The peptide sequences investigated in this work (Table 3.1) were synthesized by The

University of North Carolina Peptide Synthesis Facility, purified by HPLC, and supplied in

lyophilized form.

Table 3.1 Peptides used for assembling gold nanoparticle conjugates.

No. Peptide sequence Origin of peptide MW pKI

I CGGFSTSLRARKA Adenoviral NLS 1353 12.3

II CKKKKKKSEDEYPYVPN Adenoviral RME 2084 9.3

3.2.2 Synthesis of PEG-SH molecules

Average molecular weight 5,000 g/mol thiolated mPEG was purchased from Fluka

(PEG 5000). Synthesis of average 900 g/mol and 1,500 g/mol ethylene glycols (PEG 900 and

PEG 1500, respectively) was accomplished following procedures reported by Tremel10

(Figure 3.1).

TsCl
HO CH2CH2O H HO CH2CH2O Ts
n CH2Cl2 n
1 2

LiBr
HO CH2CH2O Br
Acetone n-1
3
O

EtOH
HO CH2CH2O S
n-1
4

HCl
HO CH2CH2O SH
n-1
5

Figure 3.1 Synthesis of PEG 900 (n = 20) and PEG 1500 (n = 34) molecules.

68
Synthesis of p-Tolylsulfonyl polyethylene glycol (2): 0.05 mols of polyethylene glycol and

an equal molar amount of triethylamine (5.06 g) were dissolved in methylene chloride. To

this solution, 9.5 g (0.05 mols) of toluene-p-sulfonyl chloride in methylene chloride were

added dropwise. The mixture was stirred overnight at room temperature. The white

precipitate of triethylamine hydrochloride was filtered off and washed with methylene

chloride. The solution was evaporated to dryness and the residue chromatographed on silica

gel using a 10:3 ratio of chloroform and acetone. Yield: 48.2% (n = 20), 58.4% (n = 34). 1H

NMR 7.78-7.76 (d, 2 H, aromatic), 7.34-7.20 (d, 2 H, aromatic), 4.15-4.12 (t, 2 H,

O2SOCH2), 3.68-3.56 (m, OCH2), 2.68 (s, 1 H, -CH2OH), and 2.43 (s, 3 H, -CH3).

Synthesis of Bromo-polyethylene glycol (3): 17 g (0.2 mols) of LiBr were dissolved

in reagent grade acetone. 21 mmols of tosylated alcohol (2) was added and the resulting

mixture stirred for 5 h at 80 oC. The mixture was then cooled to room temperature and

stirred overnight. The solvent was evaporated and chloroform added to the residue. The

white precipitate that formed was filtered off and the solution was washed twice with de-

ionized water. The organic layer was dried over Na2SO4 and the solvent was evaporated.

Yield: 60.5% (n = 20), 51.0% (n = 34). 1H NMR 3.75 (t, 2 H, CH2Br), 3.68-3.56 (m, OCH2),

3.49 (t, 2 H, CH2OH), 2.68 (s, 1 H, CH2OH).

Synthesis of thioacetate (TA) protected polyethylene glycol (4): 0.01 mols of

bromonated PEG (3) were combined with 1.78 g of potassium thioacetate in a 1:1.2 molar

ratio in ethanol. The mixture was stirred under reflux at 80 oC for 5 h. The white KBr

precipitate was filtered off and the solvent evaporated. The residue was dissolved in

chloroform and washed twice with de-ionized water. The organic layer was dried over

69
 1
Na2SO4 and the solvent was evaporated. Yield: 85.4% (n = 20), 90.2% (n =34). H NMR

2.38 (s, 3 H, CH3), 3.78-3.46 (m, CH2O), 2.67 (s, 1 H, CH2OH).

Synthesis of mercaptopolyethylene glycol (5): 1 mmol of compound (4) was

dissolved in 5 mL of 1 M solution of hydrochloric acid. The mixture was heated under reflux

for 2 h. The solvent was evaporated and the residue dissolved in chloroform. The resulting

mixture was washed twice with de-ionized water and the organic phase dried over Na2SO4.

Yield: 62.7% (n = 20), 50.4% (n = 34). 1H NMR: 3.74-3.58 (m, CH2O), 3.23-3.20 (t, 2 H,

CH2SH), 2.68 (s, 1 H, CH2OH), 2.16 (s, 1 H, CH2SH); IR 3508 (-OH), 2900-2850 (-CH2),

1454 (CH2), 1200-1100 cm-1 (C-O-C).

3.2.3 Preparation of pegylated gold nanoparticles

To prepare PEG 5000 modified gold nanoparticles, 145 µL of aqueous PEG 5000 (20

µΜ) was added to 1 mL of 20 nm citrate-coated gold nanoparticles (1.2 nM), producing a

molar ratio of PEG to gold nanoparticles of 2500:1. The mixture was stirred and incubated at

room temperature for 1h to allow complete exchange of the citrate with thiol. The mixture

was then purified by centrifugation at 13,500 g for 20 minutes. The supernatant was decanted

and the pellet was re-suspended in 1 mL PBS buffer. All other pegylated nanoparticles were

prepared in a similar fashion except that different input ratios of PEG molecules to gold

nanoparticles were used to adjust surface concentration as desired.

3.2.4 Preparation of gold nanoparticles with mixed monolayers of PEG and peptides

Two methods were evaluated for preparing mixed monolayers of PEG and peptides

on gold nanoparticles: parallel and sequential adsorption methods. In the parallel adsorption

70
method, the desired PEG and peptide were mixed with gold nanoparticles in a molar ratio of

2500:1 (total thiol: particle). For example, 72 µL of 20 µΜ PEG in PBS buffer and 72 µL of

20 µΜ cysteine-terminated peptide in PBS were combined and added to 1 mL of 20 nm

diameter citrate-capped gold nanoparticles (1.2 nM). The resulting mixture was stirred at

room temperature for 1 h to allow complete exchange of citrate with thiol on the particle

surface. The complex was washed and centrifuged twice at 11,600 g for 30 minutes. The

supernatant was decanted and the pellet was resuspended in 1 mL of PBS.

In the sequential adsorption method, PEG thiol was added to a suspension of

nanoparticles first, followed by addition of peptide. For example, 250 µL of citrate-capped

gold nanoparticles (9.4 nM, 10 nm in diameter) were mixed with 12 µL of PEG 900 (100

µM) at a ratio of PEG to gold nanoparticles of 500:1. The excess PEG molecules were

removed by centrifugation at 13,000 g for 15 min. The resulting pegylated gold nanoparticles

were then resuspended in 250 µL of PBS buffer. 23.5 µL of peptide (100 µM) was added to

produce a molar ratio of peptides to nanoparticles of 1000: 1. The mixtures were incubated

for 8 hours and purified by centrifugation at 13,000 g for 20 min and washed 2X with PBS

buffer. The final pellet was resuspended in 250 µL of PBS buffer. UV-visible absorption

spectra were then acquired to verify that the nanoparticles did not aggregate during

modification, and to determine the final concentrations of the nanoparticles.

3.2.5 Determination of critical coagulation concentrations (CCC)

To determine the critical coagulation concentration (CCC) of PEG- and peptide-

modified gold nanoparticles, a series of eppendorf centrifuge tubes containing 500 µL of

71
identical concentrations of gold nanoparticles (1.2 nM for 20nm diameter and 9.4 nM for 10

nM) were prepared. Increasing volumes of a 1.7 M NaCl stock solution were added to each

sample with the final salt concentration of ranging from 20 mM to 1 M. The tubes were

vortexed and then allowed to incubate for at least 3 hrs. Aggregation was assessed by

monitoring changes in the characteristic gold nanoparticle plasmon frequency. The threshold

NaCl salt concentration in the gold sol, which caused the rapid aggregation of the particles,

was determined as the CCC.

3.2.6 Quantifying peptide adsorption onto gold nanoparticles by fluorescence

spectroscopy

Gold nanoparticles with mixed monolayers of PEG and peptides were prepared using

the parallel or sequential adsorption methods and resuspended in 1 mL of PBS buffer. 330

µL of 0.2 mM of aqueous KCN [Handle with caution. Do not expose to acidic solution.

Ingesting or breathing CN1- can cause death.] was added to oxidatively etch the gold

nanoparticles and release the adsorbed monolayer11. Complete dissolution of the

nanoparticles was indicated by monitoring the disappearance of the gold particle plasmon

resonance absorption band at ca. 520 nm. Fluorescence spectra were acquired on the

resulting solution, and the average number of peptides per particle was obtained by dividing

the measured peptide concentration by the gold nanoparticle concentration.

3.2.7 Characterization of protein non-specific binding using fluorescence spectroscopy

Gold nanoparticles modified with PEGs or mixed monolayers of PEGs and peptides

were prepared following the procedures described above. 58 µL of fluorescein

72
isothiocyanate (FITC)-modified IgM (10.0 µM in PBS) or 38 µL of rhodamine (Rh)-labeled

BSA (15 µM in PBS) were then mixed with 0.1 mL of modified gold nanoparticles (11.6

nM) to produce a molar ratio of protein to gold particles of 500:1. The mixtures were

incubated at room temperature for 30 min and then centrifuged at 13,000 g for 15 min. The

supernatant, containing free protein, was gently removed and the remaining pellet was

washed 3X with 500 µL of wash buffer (1% SDS, 10 mM NaH2PO4, 150 mM NaCl, 1 mM

EDTA, pH 7.4). The purified gold nanoparticle pellets were resuspended in 150 µL of PBS

buffer and 50 µL of KCN solution (0.2 mM) was used to dissolve the gold nanoparticles.

Fluorescence measurements were performed on a) a blank: the cyanide-induced dissolution

of PEG or PEG + peptide modified gold nanoparticles; b) the control: the cyanide-induced

dissolution of citrate-modified gold nanoparticles fully-coated with protein; c) the sample:

the cyanide-induced dissolution of PEG or PEG + peptide modified gold nanoparticles

incubated with proteins. Protein standards were prepared by adding known amounts of

FITC-IgM or Rh-BSA to solutions of citrate-stabilized gold nanoparticles in PBS, which had

been oxidatively converted to soluble [Au(CN)4]1- by KCN. The calibration curve was used

to determine the amount of protein pelleted with, and thus assumed to be non-specifically

adsorbed to, a given sample of pegylated gold nanoparticles. Finally, the average number of

protein molecules per particle was obtained by dividing the measured protein molar

concentration by the gold nanoparticle concentration.

3.2.8 Cell treatment with gold nanoparticles

The HeLa cell line (obtained from ATCC, Rockville, MD) was maintained in EMEM

medium containing 10% Fetal Bovine Serum (FBS) and 1% antibiotics at 37°C in controlled

73
5 % CO2 atmosphere. Cells were plated on glass cover slips and grown to ~ 80 % confluency

in 12-well plates and then incubated with nanoparticles (10 nm in diameter) for 6 hours. Two

nanoparticle controls were chosen: gold nanoparticles modified with PEG 900 with input

PEG 900:particle mol ratio of 500:1, and particles modified with PEG 5000 with input PEG

5000:particle mol ratio of 250:1. Nanoparticle samples were prepared using the sequential

method. Pegylated particles were first prepared using the same PEG: particle ratios as the

two control particles. Peptides were then incubated with the particles with input

peptide:particle mol ratios of 1000:1 for an additional 8 hrs. The particles were purified by

centrifugation and the pellet was resuspended in 1 mL of PBS buffer to produce a final

concentration of 1.2 nM of gold nanoparticles. The particles were then diluted with EMEM,

producing a final particle concentration of ~120 pmol in 1.5 mL of cell media when

incubated with cells.

3.2.9 Sample preparation for inductively coupled plasma optical emission spectroscopy

(ICP-OES)

After 6 hours incubation of cells in media containing gold nanoparticles, the cover

slips were washed 3X with PBS, removed from the culture plate, and allowed to dry in air.

Dried cover slips were placed in a new well plate and 500 µL of freshly prepared Optima

grade aqua regia was added to each well. [Caution: Aqua regia is a strong acid.] The sample

remained bathed in aqua regia for 3 hours. 400 µL of the acid-dissolved samples were placed

into individual 15 mL conical tubes containing 3.5 mL of ultrapure water. To quantify the

amount of nanoparticle adsorption to the outer cell membrane, control experiments were

performed in which cells were treated and washed as described above, but with an additional

74
heparin wash step (5 U/ mL). ICP-OES analysis of the samples was then conducted on a

Perkin Elmer Optima 2100DV Optical Emission Spectrometer with a plasma flow of 18

L/min. All standards were made from 100 mg/mL SpexCertiPrep stock solution (Lot # CL3-

19AU).

3.3 Results

The objectives of this work were to determine the optimum conditions for

assembling stable peptide/PEG gold nanoparticle conjugates, and to formulate an empirical

understanding of how PEG length and peptide coverage influence cellular internalization

efficiency. In total, three different thiol-modified PEGs, four particle diameters (10 nm, 20

nm, 30 nm and 60 nm), and two peptides (Nuclear Localization Signal (NLS) and Receptor

Mediated Endocytosis (RME)) were explored. The peptides were derived from the

adenovirus fiber protein and have been used in previous cell targeting studies5, 8, 9 . The

peptides were selected because of their demonstrated ability to induce receptor-mediated

endocytosis (RME) and nuclear uptake (NLS) in a variety of cell lines when conjugated to

gold nanoparticles through bovine serum albumin linkers8, 9. However, these two peptides

have significantly different overall charge, which results in different degrees of

destabilization when conjugated to nanoparticles. PEG was chosen because it is a

biocompatible material that can improve colloid stability and resistance to protein non-

specific binding.

75
3.3.1 Assembly of PEG-coated gold nanoparticles

Of initial interest was how PEG length affects the stability of gold nanoparticles in

high ionic strength media. Two thiolated PEG molecules were synthesized, which differed in

molecular weight (MW) from 900 g/mol to 1500 g/mol (Table 3.1). A commercial PEG with

average MW 5000 was also studied.

Table 3.2 Average molecular weights and ethylene glycol (EG) units of the PEGs
investigated in this study.

MW (g mol-1) 900 1500 5000

EG Units 20 34 114

Gel electrophoresis (1 % agarose) was used to qualitatively confirm binding of PEGs

to gold nanoparticles. Figure 3.2 (left) shows a gel image of particles incubated with PEG

5000. The number labeling each lane is the molar ratio of PEG 5000 to gold nanoparticles

input into the reaction mixture. The gel shift increased with increasing PEG

5000:nanoparticle mol ratios until a plateau was reached between a ratio of 1000 and 2500.

Interestingly, the PEG 5000 particles were observed to migrate towards the negative

terminal, indicating a net positive charge. Figure 3.2 (right) shows a gel image of PEG 900-

modified nanoparticles. The gel shift decreased with increasing PEG:particle mol ratios, until

a plateau was reached at a ratio of 10,000. In contrast to particles modified with PEG 5000,

the direction of motion was towards the positive terminal, indicating a net negative charge.

A similar saturation and migration behavior was observed with PEG 1500-capped particles.

76
 (+) (-)

Figure 3.2 1% Agarose gel electrophoresis of 20 nm diameter gold nanoparticles following

the addition of different ratios of PEG 5000 (left) and PEG 900 (right). The number below
each lane indicates the added mol ratio of PEG:gold nanoparticles. Arrow indicates migration
direction.

The ζ-potential of the PEG 5000 and PEG 900 modified nanoparticles was measured

in an attempt to better understand the electrophoresis results observed in Figure 3.2. The ζ-

potential measurements showed heterogeneity in that multiple peaks were observed in the

data for both PEG conjugates (Figure 3.3). The two major peaks for each PEG molecular

weight appeared near ca. 0 mV and -50 mV. Thus, although the two nanoparticle types

appear to have opposite charges by gel electrophoresis, very little difference in their overall

charge profiles was observed in ζ-potential measurements.

77
Figure 3.3 Two data sets measuring the z-potential of PEG 5000 and PEG 900 modified 20
nm diameter gold nanoparticles.

78
 Dynamic light scattering (DLS) was also used to qualitatively confirm PEG binding.

DLS studies of the 20 nm nanoparticles revealed an increase in hydrodynamic diameter as

PEG chain length was increased as expected for monolayer formation (Table 3.3).

Table 3.3 Dynamic Light Scattering (DLS) of 20 nm diameter gold particles modified with
the indicated monolayer.

Monolayer Hydrodynamic diameter (nm)

Citrate 21.8 ± 0.5

PEG 900 29.0 ± 0.8

PEG 1500 31.3 ± 1.2

PEG 5000 40.0 ± 0.8

79
Figure 3.4 UV-visible absorption spectra of 20 nm diameter citrate-stabilized gold
nanoparticles incubated with varying concentrations of NaCl. The CCC is indicated by a
large decrease and/or redshift of the ca. 525 nm plasmon absorption band. This is also
accompanied by an obvious color change from red to purple. From these spectra, a CCC of
30 mM was determined.

Figure 3.5 UV-vis absorption spectra of PEG 5000 stabilized gold nanoparticles (20 nm in
diameter) in 500 mM NaCl solution (final concentration). The added mol ratio of PEG 5000
to gold was shown in the spectra.

80
Figure 3.6 CCC of pegylated gold nanoparticles (10nm in diameter).

81
 Applications of nanoparticles in biology require that they are stable in solutions

containing high concentrations of proteins and salts. Gold nanoparticles, however, are highly

polarizable metals with a large Hamaker constant. They are prone to aggregation in high

ionic strength solutions in which the van der Waals attraction outweighs the electrostatic

and/or steric repulsion provided by surface-bound ligands (e.g., citrate or PEG). To

determine the stability afforded by PEG ligands, critical coagulation concentrations (CCC)

were measured as a function of PEG length, nanoparticle diameter, and the number of PEG

molecules added per gold nanoparticle. Figure 3.6 shows how the CCC was determined as

citrate-coated 20 nm diameter gold nanoparticles as an example. The data from Figure 3.6

and Table 3.4 show that the CCC of gold nanoparticles depends sensitively upon all of these

parameters. Two stability trends are particularly noteworthy from Table 3.4: (i) for any given

PEG length, as the nanoparticle diameter increases, the amount of PEG that must be input

into the reaction mixture per nanoparticle increases, and (ii) for any given nanoparticle

diameter, as the PEG length decreases, the amount of PEG that must be input into the

reaction mixture increases. (This is true despite the fact that any excess PEG was removed

prior to performing the CCC tests.) These trends provide useful cues in the construction of

stable mixed PEG/peptide gold nanoparticle conjugates (see below).

82
Table 3.4 Minimum number of PEG molecules that must be added into the reaction mixture
per gold nanoparticle in order to form a stable gold sol in 1 M NaCl.

# of PEG Molecules Required per Nanoparticle

PEG MW
10 nm Diameter 20 nm Diameter 30 nm Diameter

900 500 1000 2500

1500 350 800 2000

5000 100 300 500

3.3.2 Non-specific binding of proteins to PEG-modified gold nanoparticles

In addition to improving the stability of gold nanoparticles in high ionic strength

media, one potential advantage of PEG monolayers is the prevention of protein adsorption.

Indeed, Huang and coworkers reported that PEG 150 (EG3-SH) prevented the non-specific

binding of proteins such as BSA and lysozyme to gold nanoparticles12-14. Our own gel

electrophoresis experiments performed as a control were in accord with those reported

previously, that is, gel shifts were not observed between PEG-modified gold nanoparticles

incubated in solutions containing BSA or IgM vs. protein-free solutions. However, due to

the large mass of the gold nanoparticles, gel electrophoresis is not a sensitive method for

detecting proteins adsorbed to nanoparticles. Thus a fluorescence assay was developed in

order to detect protein non-specific binding. Indeed, the assay was able to detect BSA or

IgM bound to PEG-modified gold nanoparticles, albeit in low amounts (Table 3.5). To

83
 explore a possible correlation between protein non-specific binding and particle surface

area or radius of curvature, four particle diameters were explored. It was found that the

number of proteins adsorbed per particle was particle-size dependent for diameters between

10 nm and 60 nm while the adsorbed proteins per unit of surface area was invariant or

decreased slightly.

Table 3.5 Nonspecific binding of proteins to PEG 5000-modified gold nanoparticles.

Diameter (nm) # BSA/Particle # BSA/µm2 # IgM/Particle # IgM/µm2

10 2±1 6 x 103 3±1 9 x 103

20 7±1 6 x 103 9±2 7 x 103

30 9±3 6 x 103 10 ± 4 6 x 103

60 40 ± 8 3 x 103 50 ± 6 4 x 103

3.3.3 Assembly of mixed PEG/Peptide monolayers on gold nanoparticles.

Having synthesized and characterized pure PEG monolayers on gold nanoparticles,

the co-adsorption of PEGs and cell-targeting peptides was investigated. UV-visible

absorption spectroscopy and gel electrophoresis confirmed the assembly of particles with

mixed monolayers. For example, Figure 3.7 (left) shows visible spectra of nanoparticles

prior to and following modification with a mixed monolayer of PEG 5000 and the RME

peptide. The gold nanoparticle plasmon band was shifted by only 4 nm, indicating particle

stability following formation of the mixed monolayer. Figure 3.7 (right) shows the image

from agarose gel electrophoresis. The first lane from the left contains particles modified

84
 with PEG 5000 alone. The second lane contains particles modified with PEG 5000 and the

RME peptide. The gel shift clearly confirms the formation of a mixed PEG 5000 and RME

peptide monolayer.

Figure 3.7 UV-visible absorption spectra of 10 nm diameter gold nanoparticles modified with
PEG 5000 and PEG 5000 + RME peptide (left). Agarose gel electrophoresis of gold
nanoparticles (right): Left lane contains PEG 5000-modified gold nanoparticles and right
lane contains PEG 5000 + RME peptide-modified gold nanoparticles. The particles were
prepared by sequential method.

Fluorescence spectroscopy was employed to quantify peptide loading. Figure 3.8 is

a plot of the number of rhodamine-labeled RME peptides adsorbed per particle after 6 hours

vs. the total number of RME + PEG thiols added to the reaction mixture per particle (added

at 1:1 mole ratio). The binding curves are indicative of monolayer adsorption, reaching a

plateau at approximately 600 RME peptides per 10 nm diameter nanoparticle when PEG

5000 was the co-adsorbate. Figure 3.9 shows a similar adsorption isotherm for

nanoparticles prepared by the sequential addition method.

85
Figure 3.8 Adsorption isotherm of rhodamine-labeled RME peptide adsorbed onto 10 nm
diameter gold nanoparticles. Particles were prepared using the parallel adsorption method. A
total thiol to gold nanoparticle mol ratio of 2500:1 was used with an added mol ratio of PEG
5000 to RME of 1:1.

86
Figure 3.9 Adsorption isotherms of rhodamine-labeled RME peptide adsorbed onto 20 nm
diameter gold nanoparticles. Particles were prepared using the sequential adsorption method.
The particles were pegylated by PEG 5000 (top) and PEG 900 (bottom) using added mol
ratios of PEG to particles of 300:1 and 1000:1 respectively, followed by the addition of RME.

87
Table 3.6 CCC of 20 nm diameter gold nanoparticles modified with mixed monolayers of
PEG 5000 and the NLS peptide. The conjugates were prepared using the parallel adsorption
method and a total thiol to gold nanoparticle mol ratio of 2500:1.

PEG: NLS Mol CCC (M)

Ratio

Pure NLS < 0.050

2:8 0.15

3:7 0.20

4:6 >1.0

5:5 >1.0

7:3 >1.0

We have previously observed that the addition of certain peptides to gold

nanoparticles can significantly decrease their stabilities in salt solutions15. It was thus of

interest to determine CCC values for nanoparticles modified with mixed PEG/peptide

monolayers. Table 3.6 shows the stability of nanoparticles modified with mixed monolayers

of PEG 5000 and the NLS peptide. The data show that at a constant ratio of total mols of

thiol added per mol of gold nanoparticles, increasing the mol ratio of PEG to NLS increased

nanoparticle stability. These results motivated us to compare particle stabilities for the two

ligand addition methods: parallel vs. sequential addition. Mixed PEG/RME monolayers

prepared using the parallel thiol addition strategy showed that the larger molecular weight

PEG 5000 was able to stabilize gold nanoparticles (10 nm and 20 nm diameter) in 1 M salt

88
 solutions, while the lower molecular weight PEG 900 was not (Table 3.7). In contrast,

mixed monolayers prepared by first adding the PEG followed by the RME peptide were

stable for both PEGs studied.

Table 3.7 CCC of 10 nm diameter gold nanoparticles modified with mixed monolayers of
PEG 900 and the RME peptide. The total thiol: particle mol ratio was 5000:1 for each
method.

Parallel Addition Method Sequential Addition Method*

PEG: RME Mol Ratio CCC (M NaCl) PEG: RME Mol Ratio CCC (M NaCl)

7:3 0.2 7:3 >1.0

5:5 X 5:5 >1.0

3:7 X 3:7 >1.0

X Indicates aggregation immediately upon addition of peptide.

* PEG added first followed by peptide.

It was shown above that approximately 7% of the surface of a pegylated 20 nm

diameter gold nanoparticle was covered by non-specifically adsorbed BSA molecules. It was

thus of interest to determine whether particles functionalized with PEG and peptides could

resist protein nonspecific binding as well. This was accomplished by preparing mixed

PEG/peptide monolayers of varying mol fraction and incubating them with BSA (Figure

3.10). The amount of non-specifically bound BSA was found to be low and relatively

independent of PEG mol fraction and PEG molecular weight (Table 3.8)

89
Figure 3.10 Nonspecific binding of BSA to (PEG 5000 + RME peptide)-coated 20 nm
diameter gold nanoparticles (top) and (PEG 900 + RME peptide)-coated gold nanoparticles
(bottom). Particles were prepared using the sequential adsorption method.

90
Table 3.8 Nonspecific binding of protein BSA to 20 nm diameter gold nanoparticles
modified with the indicated monolayer.

Monolayer # BSA/particle*

(PEG 5000 + RME) 11 ± 1

(PEG 900 + RME) 8±1

* Average taken over all PEG:RME mol fractions studied.

From the preceding studies, it follows that gold nanoparticle sols containing mixed

PEG/peptide monolayers can be assembled, and that their stability towards aggregation in

high ionic strength media depends upon PEG length. Longer PEGs afford enhanced stability

over shorter ones. The function of the targeting peptides was then tested by incubating

nanoparticles modified with the RME peptide and either PEG 900 or 5000 with HeLa cells

and quantifying cellular internalization using ICP-OES spectroscopy. These studies were

designed to determine whether the longer PEG molecules sterically hinder the targeting

peptides, thus preventing them from interacting with their target receptors on the cell

membrane.

91
Figure 3.11 ICP-OES analysis of HeLa cells following 6 hours incubation with media
containing 10 nm diameter gold particles modified with mixed PEG/RME monolayers.
Samples were prepared as follows. (A) PEG 900 to particle mol ratio of 500:1. (B) PEG 900
to particle mol ratio of 500:1 followed by addition of peptide with a mol ratio of 1000:1. (C)
PEG 5000 to particle mol ratio of 250:1. (D) PEG 5000 to particle mol ratio of 250:1
followed by the addition of peptide with a mol ratio of 1000:1.

Figure 3.11 shows the number of 10 nm diameter nanoparticles per well for the mixed

monolayer formulations PEG 900 + RME and PEG 5000 + RME prepared using the

sequential adsorption method. Both particle formulations were able to enter HeLa cells in 6

hours. Surprisingly, particles modified with mixed monolayers of PEG 5000 and the RME

peptide were found inside of cells in higher abundance compared to particles modified with

PEG 900 and the RME peptide. Control experiments performed on nanoparticles modified

with either PEG alone showed comparably less cellular internalization. In an attempt to

deconvolute the amount of gold internalized from the amount that may have been non-

specifically adsorbed to the outer cell membrane, control experiments were performed in

92
which the cells were washed with heparin sulfate. The excess heparin was expected to

compete effectively with any peptide-gold nanoparticle conjugates adsorbed electrostatically

to the outer surface of the cell membrane. Despite this treatment, the total gold observed via

ICP-OES was unaffected, suggesting that nanoparticle adsorption to the cell membrane does

not contribute significantly to the total gold measured by ICP-OES.

3.4 Discussion

Gold nanoparticles have shown great promise as therapeutics, therapeutic delivery

vectors, and intracellular imaging agents7-9, 16-18. For many biomedical applications, selective

cell and nuclear targeting is desirable, and this remains a significant practical challenge in the

use of nanoparticles in vivo. This challenge is being addressed by the incorporation of cell-

targeting peptides or antibodies onto the nanoparticle surface. As these ligands can affect the

size, charge, and stability of the resulting bioconjugates, we view studies of monolayer

composition-nanoparticle function relationships as being critical to the development of gold

nanoparticles that can interface effectively with biological systems.

The systematic studies reported here revealed important differences in the stabilities

of gold nanoparticles modified with PEG and mixed PEG/peptide monolayers. We first

considered nanoparticles modified solely with PEG monolayers. Gel electrophoresis, DLS,

and CCC assays all qualitatively confirmed conjugation of PEGs to gold nanoparticles. Gel

electrophoresis revealed opposing migration directions for nanoparticles modified with PEG

900 vs. PEG 5000, suggesting that under the conditions in which the gels were run the charge

of the two constructs is different, PEG 5000-particles being positively charged. These

observations may be due to differences in either the displacement of citrate molecules or the

93
sequestration of cations by the two PEGs. In contrast to the gels, ζ-potential measurements

revealed little difference in charge of the two conjugates, although sample heterogeneity

made any quantitative conclusions difficult. Thus, while gel shift assays are useful in

determining qualitatively the adsorption of PEGs and peptides to gold nanoparticles, the

apparent difference in surface potential of these conjugates remains an interesting and

important area for further study, particularly insofar as particle charge could influence

translocation across cell membranes.

CCC data showed that nanoparticle stability in high ionic strength media depends

upon PEG length, nanoparticle diameter, and the ratio of PEG molecules:nanoparticles input

into the reaction mixture. Longer PEGs, smaller nanoparticles, and larger PEG:nanoparticle

ratios favor more stable sols. The sigmoidal CCC curves reported in Figure 3 suggest that a

threshold coverage of PEG must be reached before stability begins to increase. These

observations are in accord with DLVO theory, which predicts greater stability for smaller

nanoparticles because the van der Waals attraction energy is minimized. Flory-Krigbaum

theory also indicates that longer surface-bound ligands and higher ligand coverage will favor

sol stability because of increased steric repulsion19, 20.

The diameter of biomolecule-nanoparticle conjugates is yet another important design

parameter, particularly when access to the cell nucleus is desired. The diameter of the nuclear

pore complex in HeLa cell lines, is ca. 39 nm21. DLS measurements showed that PEGs 900,

1500, and 5000 add 3.5 nm, 5 nm, and 9 nm to the hydrodynamic radius of a gold

nanoparticle, respectively. Thus, while PEG 5000 can offer increased stability to 20 nm

diameter gold nanoparticles, it could also compromise nuclear translocation efficiency

because of its added bulk.

94
 We have also considered non-specific protein adsorption to PEG-modified gold

nanoparticles. The ability of PEG to block proteins from adsorbing onto surfaces has been

studied extensively, and it was reported recently that this property translates to PEG-

modified gold nanoparticles. In accord with prior results12, 13, adsorption of PEG-modified

gold nanoparticles was not observed using a gel-shift assay. However, a more sensitive

fluorescence assay was able to detect the adsorption of BSA and IgM to PEG-modified gold

nanoparticles. The measured 7 ± 1 BSAs per 20 nm diameter nanoparticle can be compared

to the maximum number of BSA proteins that can be accommodated on a 20 nm diameter

citrate-modified gold nanoparticle, approximately 100 per nanoparticle15. While the absolute

number of adsorbed BSA and IgM proteins increased as particle diameter was increased, the

number of proteins per unit area remained relatively constant. Thus, within the size regime

studied here, there are little inherent differences in particle surface morphology or PEG

packing that influence non-specific protein adsorption.

One of the most compelling attributes of gold nanoparticles is the ease with which

mixed thiol monolayers can be assembled on their surfaces22. The ability to create mixed

monolayers on a nanoscale platform provides a powerful tool that can be used to improve

water solubility, tune sterics, and control the trajectory of nanoparticles in cells. Of interest

here was whether the addition of PEG as a co-adsorbate could increase the solubility of gold

nanoparticles modified with certain cell-targeting peptides (NLS and RME, Table 3.1).

Mixed peptide/PEG monolayer formation was characterized with gel electrophoresis,

fluorescence spectroscopy, and CCC assays. Two methods of monolayer formation were

investigated, which differed only in the order in which the thiols were added to gold

nanoparticles. Insofar as stability vs. aggregation in salt solutions is concerned, nanoparticles

95
prepared using the sequential adsorption method were superior to those prepared by the

parallel adsorption route. For example, Table 3.7 shows that nanoparticles prepared by

incubating a gold sol in a solution containing a 1:1 mol ratio of PEG 900 and the RME

peptide aggregated rapidly, even without adding excess salt. In contrast, nanoparticles

prepared by adding PEG 900 to the gold sol first, followed by addition of the RME peptide,

resisted aggregation in solutions containing NaCl at concentrations in excess of 1 M. We

suspect that the origin of these marked differences in stability is kinetic. The highly cationic,

lysine rich RME peptide may associate with the anionic gold nanoparticles more rapidly than

PEG thiols. As a consequence, the charge on the nanoparticles would be neutralized prior to

the formation of a PEG steric barrier. This would be expected to result in nanoparticle

aggregation. When even a submonolayer of PEG is attached to the nanoparticle first,

however, enough steric repulsion could be created to prevent aggregation upon peptide

addition.

Finally, the ability of nanoparticles modified with mixed PEG/RME peptide

monolayers to enter HeLa cells was investigated. Given the relative lengths of PEG 5000 and

RME peptide, we initially hypothesized that when these two ligands are bound to a

nanoparticle, access of the RME signal to its membrane-bound target would be significantly

hindered. Based upon this simple steric argument we expected less cellular internalization of

PEG 5000/RME conjugates vs. the corresponding conjugates prepared with PEG 900. ICP-

OES analysis of cell uptake showed, however, that the number of PEG 5000/RME

conjugates inside HeLa cells after 6 hours was nearly a factor of 4 larger than PEG 900/RME

conjugates. Even accounting for slight differences in the total number of peptides per

nanoparticle (150 RME for PEG 5000/RME conjugates vs. 100 for PEG 900/RME

96
conjugates according to their respective isotherms), PEG 5000/RME conjugates enter HeLa

cells with surprising efficiency compared to PEG 900/RME conjugates. This could be a

manifestation of the larger footprint (lower packing density) of PEG 5000 on a gold surface,

relative to PEG 900, a possibility we have attempted to address using a Gaussian chain model

for PEG packing on a gold surface.

Figure 3.12 side and top projections of Gaussian chain on a surface

We can consider the Gaussian chain model on a surface as consisting of a vector

defined by d that rotates in a cylindrical fashion about a fixed point. In the present model,

the fixed point is defined by the gold-thiolate bond. Figure 3.12 shows side and top

97
projections. The circular projection shown in the right-hand panel of the figure implies that

there is a void space of approximately 10% on the surface. This is illustrated in Figure 3.13.

Figure 3.13 Circular projection of Gaussian chain

Assuming the PEG molecules can be modeled as non-entangled Gaussian chains, the

average diameter of each PEG is d = L√N, where L is the distance between each successive

monomer and N is the number of ethylene glycol monomers (-O-CH2-CH2-). The distance L

is approximately 4.25 Å. For PEGs 900 and 5000 there are 20 and 110 monomer units,

respectively. The Gaussian chain lengths for PEGs 900 and 5000 are estimated to be d = 1.9

nm and 4.5 nm, respectively. The surface area of a 20 nm particle is ~1250 nm2. Assuming

that the PEGs bind to the surface such that they sweep out a cone with a tilt angle of

98
approximately 45o, the footprint of each of the PEGs on the surface of the nanoparticle can

be estimated to be approximately equal to πr2 where r = d/√2. Therefore, the area covered by

the respective PEGs is given by πd2/2 so that PEGs 900 and 5000 cover ~6 nm2 and ~32 nm2,

respectively. If we assume complete coverage this implies that the number of PEGs that can

bind is ~190 and ~36 for PEG 900 and 5000, respectively. For comparison, fully extended

chains would be ~8.6 nm and ~48.2 nm, for PEG 900 and PEG 5000, respectively. DLS

measurements in Table 3 show that PEG 900 and PEG 5000 add 3.5 nm 9 nm, respectively,

to the radius of the particles. These values are in reasonable agreement with the Gaussian

chain model, but indicate that the chains are constrained on the surface and add more to the

radius than the Gaussian chain model would predict. Nonetheless, these considerations give a

rough estimate of the substantial differences between the different PEG lengths.

There are several factors that influence the surface charge density and accessibility of

targeting peptides on PEG labeled nanoparticles. Based on the smaller size, the packing of

PEG 900 is expected to be better than that for PEG 5000. We can think about the packing of

PEGs and smaller molecules such as peptides and citrate using a “trees and grass” model. In

this model we can envision the exposure of surface groups as depending on the extent to

which the larger PEG molecules (trees) can cover the smaller molecules near the surface

(grass). The poorer packing of the larger PEG molecules likely leads to open areas that can

expose peptides for cell targeting.

We can consider the positive charge of the PEG 5000-coated nanoparticle as a second

factor that may improve cell-targeting efficiency. PEG 5000/RME conjugates appear to

contain a net positive charge, which may provide a favorable interaction with the cell

99
membrane. In contrast, the apparent net negative charge of PEG 900/RME conjugates could

hinder its interaction with the cell membrane.

3.5 Conclusions

Mixed PEG/peptide monolayers on gold nanoparticles have been synthesized and

characterized for their stability in high ionic strength media. PEG length and particle size are

two important adjustable parameters for creating stable peptide/gold nanoparticle conjugates.

Although our intuition led to the initial hypothesis that longer PEG co-adsorbates (e.g., MW

5000) would sterically hinder a shorter peptide from accessing cell membrane receptors, this

was not born out by experiment. A mixed monolayer consisting of a receptor mediated

endocytic peptide and PEG 5000 was internalized at least as efficiently as the same

conjugates with PEG 900 as co-adsorbate.

100
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(21) Pante, N.; Kann, M. Molecular Biology of the Cell 2002, 13, 425-434.

102
(22) Shon, Y. S.; Mazzitelli, C.; Murray, R. W. Langmuir 2001, 17, 7735-7741.

103
Chapter 4 Antisense Oligonucleotide Modified Nanoparticles for

Up-regulation of Luciferase Gene Expression

104
4.1 Introduction

The design, preparation, characterization and nuclear targeting of antisense

oligonucleotides (the phosphorothioate 2’-O-methyl-oligoribonucleotides, ON705 and

Peptide Nucleic Acid, PNA) modified nanoparticles are described herein.

Three bioconjugate strategies were employed to prepare antisense oligonucleotide-

modified gold nanoparticles, two of which have been described in previous sections 1.5 and

2.3. The first strategy was the use of cross linker streptavidin (SA). SA was electrostatically

bound to citrate-stabilized gold nanoparticles and linked to biotin-modified biomolecules as

well. (Figure1.7). The second was the direct attachment of thiolated antisense

oligonucleotides to nanoparticles via the ligand exchange method (Figure 2.2). The last was

involved the use of a leash, a 12-mer single strand DNA (ssDNA) (TTT TGT AAC TGA GGT)

that is partially complementary to ON705 or Peptide Nucleic Acid (PNA).

By using alternate gene splicing as a positive readout assay, as described in section

1.8, we have examined whether the presence of the nanoparticles (gold nanoparticles and

quantum dots) affects the ability of the oligonucleotides to interact with pre-mRNA target.

Five different peptides (Table 4.1) have been studied attempting to enhance the nuclear

delivery of nanoparticles. A popular transfection agent, Lipofectamine 2000 (LF) was also

used to improve the nuclear targeting ability of nanoparticle vectors.

Interesting findings are reported here: (1) The antisense oligonucleotides modified

with nanoparticles were bioavailable to pre-mRNA in the nucleus of the cells; (2) the

nanoparticles improve the nuclear targeting ability of modified oligonucleotides with LF

compared with free oligonucleotides with LF. These studies provide new insights into the

possibilities and limitations of the biological applications of multifunctional nanoparticles.

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4.2 Materials and Instrumentation

Citrate-coated gold nanoparticles (10 nm and 20 nm in diameter) were purchased

from Ted Pella. Streptavidin (SA) coated nanocrystals (CdSe@ZnS), QD605, were

purchased from Quantum Dot Corporation. Streptavidin (SA) was purchased from Pierce.

Texas Red-labeled streptavidin was purchased from Biomeda Corporation. Tris-HCl

prepacked PAGE gels, native sample buffer and biosafe Coomassie G250 stain were

purchased from Bio-Rad. Agarose DNA grade, tris-boric acid-EDTA (TBE) 5X buffer, tris-

glycine 10X buffer and 20X saline-sodium citrate solution were purchased from Fisher

Scientific. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and

Dulbecco’s Phosphate-buffered Saline (DPBS) were purchased from BioWhittaker. Opti-

MEM reduced serum medium and Lipofectamine (LF) were purchased from Invitrogen.

pLuc 705 HeLa cells were a gift from R Kole at Lingberger Comprehensive Cancer Center at

University of North Carolina at Chapel Hill. Luciferase Reporter Assay kit was purchased

from Applied Biosystem International (ABI).

The peptide sequences investigated in this work (Table 4.1) were synthesized by The

University of North Carolina Peptide Synthesis Facility, purified by HPLC, and supplied in

lyophilized form. The oligonucleotides were custom ordered from MWG-Biotech or IDT.

Peptide nucleic acid (PNA) was a gift from ABI (Table 4.2).

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Table 4.1 Peptides used in this study

# Sequence (NH2-)* Origin Ref.

1 -FNPVYPYEDESKKKKKKGG- Adenoviral RME 1, 2

2 -AKRARLSTSFGGG- Adenoviral NLS 1, 3

3 -GFMDGRGGKKKKKKG- Integrin binding domain 4, 5

4 -GGVKRKKKPEDPYGG- NLS from large T antigen of SV-40 virus 6, 7

5 -KETWWETWWTEWSQPKKKRKV- Pep1 7, 8

*All peptides have a fluorescence label at N-terminal and Biotin Nova Tag at C-terminal.

Table 4.2 Oligonucleotides used in this study

Name Sequence (5’~3’)

PNA CCT-CTT-ACC-TCA-GTT-ACA-FITC

Leash thiol Thio-modifier C6 S-S TTT TGT AAC TGA GGT

Biotin-Leash Biotin TTT TGT AAC TGA GGT

ON705 CCU CUU ACC UCA GUU ACA

Thiol- ON705 Thiol MC6-D CCU CUU ACC UCA GUU ACA

Biotin-ON705 Bio TEG CCU CUU ACC UCA GUU ACA 3'

R30F Thiol-GGA GAC TGT TAT CCG CTC AGA ATT CCA CAC -Fluorescein

All UV-vis absorption measurements were acquired on a Hewlett Packard 8453

Chemstation Photo Diode Array Spectrophotometer with attached Chemstation software.

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Fluorescence intensity data were obtained using a Perkin Elmer luminescence spectrometer

LS50B or a BioTek FL-600 plate reader. Dynamic light scattering (DLS) measurements were

obtained on a Malvern Zetasizer 1000.

4.3 Experimental procedures

4.3.1 Preparation of streptavidin (SA) coated gold nanoparticles

1 mg SA was dissolved in 1 mL of 10 mM phosphate buffered saline (PBS) to

produce a 16.7 µM SA stock solution. 140 µL of the above solution was added into 500 µL

of 9.5 nM citrate capped gold nanoparticles (10 nm in diameter), producing a molar ratio of

SA to gold nanoparticle of 500:1. The mixture was vortexed and then incubated at room

temperature for at least half an hour. The excess SA was removed by centrifugation at 13000

rpm for 20 min. The pellet was re-suspended in 500 µL of PBS buffer.

4.3.2 Determination of surface coverage of Streptavidin (SA) on gold nanoparticles

In order to obtain the binding isotherm of Texas Red labeled streptavidin (TR-SA) on

gold nanoparticle surfaces, different surface coverages of SA on gold nanoparticles were

prepared with a similar procedure. For example, 10 nm gold nanoparticles (500 µL of 9.5

nM) were mixed with 63 µL of TR-SA solution (18.9 µM in PBS), producing a molar ratio

Au: TR-SA of 1:250. After stirring for 30 min, those sols were centrifuged at 13000 rpm for

15 min. Supernatants containing unbound proteins were carefully removed from the pellets,

followed by washing the sediments with 200 µL of phosphate buffer two more times. All the

supernatants for each sample were collected and then diluted to 1 mL. A standard curve was

created by measuring the fluorescence intensities of TR-SA solutions ranging from 0.078 µM

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to 10 µM in 10 mM phosphate buffer (pH 7.4) and was used to determine the concentration

of unbound TR-SA in the supernatant solution. The amount of TR- SA molecules bound on

nanoparticle surface was calculated by subtracting free TR-SA from the total amount of TR-

SA added initially.

4.3.3 Annealing and confirmation of Peptide Nucleic Acid (PNA) to Leash

PNA was annealed to the leash sequence according to established procedures9, 10 with

a few modifications. 50 µL of PNA stock solution (100 µM) and 50 µL of leash stock

solution (100 µM) were mixed and diluted to 200 µL with 10mM PBS buffer. The mixture

was incubated in a water bath at 60ºC for 1 hr and cooled down naturally overnight. The

solution was maintained at 4ºC until use.

The annealing of PNA to the leash was verified by native PAGE gel electrophoresis.

Samples (5 ~10 µL), including PNA, leash, and PNA-leash duplex were diluted in the native

sample buffer in a 1:2 ratio, and loaded onto an 18% Tris-HCl native polyacrylamide gel. A

100-base pair ladder from BioRAD was used as a size marker. The gel was run in tris-glycine

1X buffer at 150V for about 1 hour. The gel was subsequently stained by biosafe Coomassie

blue according to the manufacturer’s protocol.

4.3.4 Preparation of leash-PNA duplex coated gold nanoparticles/Quantum dots (QDs)

SA-gold nanoparticle complexes were prepared as described above. Then, 28.5 µL of

the biotinylated leash-PNA duplex (25 µM) was added to 0.5 mL of the re-suspended SA-

gold nanoparticle solution (9.5 nM) to achieve a 150:1 of leash-PNA: particle ratio. The

mixtures were incubated at room temperature on a shaker for 1 hour, followed by

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centrifugation at 12,000 rpm for 20 minutes to remove the excess duplex and re-suspension

of the pellets in 0.5 mL of 10 mM phosphate buffer. Biotin-leash-PNA duplex coated SA

modified QDs could be prepared with same method except that a different ratio of duplex to

QDs such as 50:1 was used.

4.3.5 Preparation of peptide-modified QDs

The peptides were incubated with SA modified QDs (~10-15nm in diameter) at molar

ratio of 10:1 to 20:1 in phosphate buffer saline (PBS) with 20 min of reaction time. The

incorporation of biotinylated peptides onto the SA-QD conjugates under these conditions is

best probed by gel electrophoresis, where the ratio of peptide/QDs spans from 1:1 to 50:1.

4.3.6 Preparation of ON705-SA-gold nanoparticles

The oligonucleotide, ON705 was incubated with 1 mL of SA coated gold

nanoparticles (10nm in diameter) at molar ratio of 75:1 in PBS. After 45 min, the mixture

was centrifuged at 13000 rpm for 20 min and the pellet was washed 2 more times with PBS.

The resulting nanoparticles were resuspended in 1 mL of PBS.

4.3.7 Preparation of (ON705 + peptides)-SA-gold nanoparticles

The peptides were mixed with ON705 in either a 1:1 or a 2:1 molar ratio before

mixing with 1mL of gold nanoparticles (10 nm in diameter). The mixtures were incubated at

room temperature for 1 hr and purified by centrifugation (2X, 13000 rpm for 20 min). The

modified nanoparticles were resuspended in 1 mL of PBS buffer.

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4.3.8 Preparation of ON705-thiol (S)- gold nanoparticles

Oligonucleotide modified gold nanoparticle conjugates were prepared as previously

11, 12
described with a few modifications . Briefly, 50 µL of a 47.5 µM of oligonucleotide

solution was added to 500 µL of 10 nm diameter gold nanoparticles (9.5 nM). The molar

ratio of the final concentration of oligonucleotides to colloid was 400:1. The samples were

placed into a water bath at 37 °C for 8 h, after which the solution was gradually brought to

0.1 M NaCl/10 mM Na phosphate pH 7.4. The samples were left in the solution for an

additional 16 h at 37 °C. Then, samples were centrifuged at 13500 rpm for 15 min three

times, with 2 times of wash using 500 µL of 0.1 M NaCl/10 mM Na phosphate pH 7.4 in

between to remove excess oligonucleotides. The final pellet was resuspended in 500 µL of 10

mM Na phosphate. Since a small amount of gold nanoparticles was discarded with the

supernatant in the washing procedure, the corresponding nanoparticle concentration was

determined by UV-visible absorption spectroscopy.

4.3.9 Determination of surface coverage of oligonucleotides on particle surface

The quantification method to determine the surface coverage of oligonucleotides on

nanoparticle surface was mainly based on the literature procedure11. Briefly, mercaptoethanol

(ME) was added (final concentration 14 mM) to gold nanoparticles (20 nm in diameter)

modified with fluorescein-labeled oligonucleotides in 10mM phosphate, 0.1 M NaCl, pH 7.4.

After 15 h incubation at room temperature with intermittent shaking, the solutions containing

displaced oligonucleotides were separated from the gold by centrifugation at 10500 rpm for

15 min three times, with 2 times of wash using 200 µL of 10 mM Na phosphate pH 7.4 in

between in order to collect all free fluorescein-oligonucleotides. Standard linear calibration

111
curves were prepared with known concentrations of fluorescein-oligonucleotides using

identical buffer and ME concentrations. The fluorescence intensities were converted to molar

concentrations of fluorescein-oligonucleotides by interpolation from a standard curve.

4.3.10 Determination of critical coagulation concentrations (CCC)

To determine the critical coagulation concentration (CCC) of SA, peptides and

oligonucleotides modified gold nanoparticles, a series of eppendorf centrifuge tubes

containing 500 µL of identical concentrations of gold nanoparticles (1.16 nM for 20nm

diameter and 9.4 nM for 10 nM) were prepared. Increasing volumes of a 1.7 M NaCl stock

solution were added to each sample with the final salt concentration ranging from 20 mM to

1 M. The tubes were vortexed and then allowed to incubate for at least 3 hrs. Aggregation

was assessed by monitoring changes in the characteristic gold nanoparticle plasmon

frequency. The threshold NaCl salt concentration in the gold sol, which caused the rapid

aggregation of the particles, is determined as the CCC.

4.3.11 Nanoparticle transfection with pLuc HeLa cells

The experimental protocols were similar to those previously described13. Briefly, The

pLuc HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)

supplemented with 10% Fetal Bovine Serum (FBS) at 37°C in a humidified atmosphere with

5% CO2. HeLa 705 cells were grown in 24-well flasks to 95% confluency. The cells were

washed twice with Dulbecco’s phosphate-buffered saline (DPBS). The control experiments

were performed with 1) mock transfected pLuc 705 cells, i.e. cells were incubated with

media only or media containing LF; 2) free oligonucleotides in the absence of LF; 3) free

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oligonucleotides in the presence of LF. Gold nanoparticles modified with ON705 were

purified in order to remove excess ON705 as described in section 4.3.6 or 4.3.8. The

nanoparticles incubated with cells were tested in two conditions as well: with or without the

presence of LF.

The samples were mixed in Opti-MEM reduced serum media. For example, a 0.1 mL

aliquot of an oligonucleotide at a given concentration (0.3 µM) in Opti-MEM was mixed

with 0.1 mL of Opti-MEM containing 4 µL of Lipofectamine. After being mixed, the

preparation was left at room temperature for 15 min, followed by dilution to 1.0 mL with

Opti-MEM. The cells were incubated at 37°C for 4 or 5 h. The final concentrations for all

oligonucleotides with or without nanoparticles were between 0.015–0.1 µM. Subsequently,

the media was replaced with DMEM containing 10% FBS. After an additional 18 h, the

luciferase reporter assay was performed following manufacturer’s protocol (ABI). All

experiments were done in triplicate.

4.4 Results

4.4.1 Assembly and characterization of (peptides+ oligonucleotides)-coated particles

Agarose gel electrophoresis (1 %) was used to confirm the binding of SA to gold

nanoparticles and the binding of peptides and oligonucleotides (ON705) to SA-gold

nanoparticles. Citrate-coated gold nanoparticles tend to aggregate in the running condition of

gel electrophoresis due to the high ionic concentration of the running buffer. The migration

of the gold nanoparticles as shown in lane 1 in figure 4.1 indicated SA stabilized the particles

and charge/mass ratio of the particles was changed. Thus, SA was confirmed bound to gold

nanoparticles. The gel shifts between lane 1 and lanes 2-5 respectively confirmed the binding

113
of ON705 to SA-coated gold nanoparticles as more ON705 was attached to particles, the

charge/mass ratio of the particles was increased. Saturation of ON705 on SA-gold

nanoparticle occurred at molar ratio of ON705: gold of roughly 500:1. Similarly, the distance

difference of band migrations revealed in figure 4.2 confirmed the binding of peptide large T

to gold nanoparticles as well as the formation of mixed monolayer of peptides and

oligonucleotides on SA-gold nanoparticles with different molar ratio of peptide to

oligonucleotides.

Figure 4.1 1% Agarose gel electrophoresis of ON705-SA-gold nanoparticles. Lane 1: SA-

gold nanoparticles with added ratio of SA: gold nanoparticle of 500:1. Lane 2-5: ON705-SA-
gold nanoparticles with increasing ratios of ON705 to gold nanoparticle of 100, 200, 300 and
500 respectively.

Figure 4.2 1% Agarose gel electrophoresis of modified 10 nm diameter gold nanoparticles.

Lane 1: SA-gold nanoparticles, Lane 2: large T-SA-gold nanoparticles, Lane 3: ON705-SA-
gold nanoparticles, Lane 4: (ON705+ large T)-SA-gold nanoparticles with 1:1 ratio of
ON705: large T, Lane 5: (ON705+BLT)-SA-gold nanoparticles with 2:1 ratio of ON705:
large T.

114
 Dynamic light scattering (DLS) also qualitatively confirmed the binding of SA and

ON705 to gold nanoparticles. These data showed an increase in the hydrodynamic diameter

(nm), which is expected upon the binding of SA/oligonucleotides/(SA+peptide)-SA to citrate

coated gold nanoparticles (Table 4.3). These results also assured that the size of the

nanoparticle conjugates was compatible with the nuclear pore complex (< 40 nm)14.

Table 4.3 Dynamic Light Scattering (DLS) of 10 nm diameter gold particles modified with
the indicated molecules.
Monolayer Hydrodynamic diameter (nm)

Citrate 10.4 ± 0.5

SA 18.4 ± 0.9

(ON705+peptides)-SA 20.0 ± 1.5

ON705-SH 20.3 ± 1.8

Fluorescence spectroscopy was employed to determine the surface coverage of SA

and thiolated-oligonucleotides on gold nanoparticle and it was found that the surface

coverage for SA was 14 ± 2 pmol/cm2 and for thiolated-oligonucleotides was 26 ± 3

pmol/cm2.

The stability of gold nanoparticles is of paramount importance in biological

applications. Electrostatically stabilized gold nanoparticles are very sensitive to electrolytes.

Cell media contains approximately 150 mM of NaCl, which can compress the ionic double

layer and thereby reduce the electrostatic repulsion between particles. When van der Waals

attraction outweighs the electrostatic and/or steric repulsion, gold nanoparticles aggregate.

Citrate-stabilized gold nanoparticles form a precipitate immediately upon the addition of

particles into cell media. This can be easily observed in a phenol-free DMEM medium by a

115
color change from red to blue. To determine the stability of the gold nanoparticles afforded

by the protein, peptide and oligonucleotides, a critical coagulation concentration (CCC) assay

was conducted. It was found that SA-gold nanoparticles were stable in solutions containing

up to 800 mM NaCl. ON705-SA-gold nanoparticles were stable in solutions up to 1 M NaCl

and thiolated ON705-gold nanoparticles were stable in solutions up to 500 mM NaCl (Table

4.4). The stability of (ON705+peptide)-gold nanoparticles varied as different types of

peptides were used. All the formulations of (ON705+peptides)-gold nanoparticles that were

studied in cell culture were stable in up to 300 nM NaCl solution.

Table 4.4 CCC of 10 nm diameter gold nanoparticle modified with SA and ON705
Monolayer on Gold particles (10nm) CCC (mM)

Citrate 50

SA 800

ON705-SA 1000

ON705-S 500

(ON705+peptide)-SA- > 300

4.4.2 Antisense activity of nanoparticles examined by luciferase assay system

The antisense activity of nanoparticles modified with oligonucleotides was studied

with the pLuc 705 HeLa cells, which have been utilized to quantitatively study the potency of

antisense agents for the alteration of gene splicing13.

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4.4.2.1 Relative antisense activity of gold nanoparticles modified solely with SA-

oligonucleotides

SA was used as both a stabilizer to electrostatically stabilize gold nanoparticles in a

physiological environment and as a bridge linker to attach biotin-modified biomolecules to

nanoparticles. This strategy affords stable gold nanoparticles with simple bioconjugate

chemistry for cellular uptake and nuclear targeting.

By performing a luciferase assay with particles modified solely with

oligonucleotides, which were transfected with LF, it was found that the oligonucleotides

were able to reach the nucleus and interact with mRNA in the splicesome (Figure 4.3 B).

Moreover, nanoparticles increased the observed level of luciferase expression in the

presence of LF. For the same concentration of ON705 in the cell medium, the luciferase

expression level could be up to 5-fold higher when ON705 was coupled to gold

nanoparticles than without nanoparticles (Figure 4.3).

4.4.2.2 Relative antisense activity of gold nanoparticles modified with both peptide and

oligonucleotides

In an effort to explore the use of peptides to target the nuclei of mammalian cells, 5

different peptides (Table 4.1) were examined with gold nanoparticles using similar

chemistry as described above. Peptides, coupled to gold nanoparticles along with ON705

oligonucleotides (Figure 4.4 as an example), were tested using the Luc expression assay.

The data showed that under the current experimental conditions, peptides did not enhance

the nuclear targeting of oligonucleotides modified with gold nanoparticles in the absence of

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 LF (Figure 4.5). However, the same formulations of gold nanoparticles did target the

nucleus of cells in the presence of LF (Figure 4.6).

Figure 4.3 Relative antisense activities of modified gold nanoparticles. Samples are as
follows: A, ON705 + LF; B, gold-SA-ON705 + LF; C gold-SA- (ON705+pep) + LF.

118
Figure 4.4 Representative examples of particle formulations with (peptide+
oligonucleotide).The numbers correspond to the formulations in figure 4.5 and 4.6.

Figure 4.5 Relative antisense activity of gold nanoparticles modified with peptide and
ON705 in the absence of LF. Formulation 1 was positive control, free ON705+ LF, 2 was
mock-transfected 705 HeLa cells. Formulations 3~8 were described in figure 5.
Concentrations of ON705, peptides, SA and gold nanoparticles were as follows respectively:
0.3 µM, 0.3 µM, 0.15 µM and 6 nM. Higher concentrations (for example, 0.6 and 1.2 µM) of
both ON705 and peptides were tested and similar results were obtained.

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Figure 4.6 Relative antisense activity of gold nanoparticles modified with peptide and
ON705 in the presence of LF. Formulation 1 was positive control, free ON705+ LF, 2 was
mock transfected 705 HeLa cells supplemented with LF. Formulations 3~8 were described in
figure 5 except that they each were combined with LF. Concentrations of ON705, peptides,
SA and gold nanoparticles were: 0.3 µM, 0.3 µM, 0.15 µM and 6 nM, respectively.

The expression of luciferase was measured for increasing concentrations of ON705

both with and without nanoparticles at a constant amount of LF (8 µg) per well. Figure 4.7

shows that the Luc level reached a maximum at 0.3 µM of ON705 and then decreased to near

background levels at 1.0 µM in agreement with a previous report13. This phenomenon has

been explained as the dependence of the charge ratio of oligonucleotide to cationic lipid. At 8

µg of LF, the optimum value was 0.3 µM. Excess oligonucleotide results in a decrease in

luciferase expression because of the charge imbalance in the LF complex13.

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Figure 4.7 Relative antisense activity of different amounts of ON705-gold and ON705 only
with 8 µg of LF in pLuc 705 HeLa cells.

Fixed concentrations of both ON705 and nanoparticles with decreasing

concentrations of LF from 4 µL to 1 µL of 2mg/mL were tested to study the dependence of

antisense activity on LF concentration in more detail. Increasing the amount of LF applied,

increased antisense activity was observed (Figure 4.8).

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Figure 4.8 Relative antisense activity of ON705-gold and ON705 only with a fixed
concentration of ON705 (0.3 µM) in pLuc 705 HeLa cells.

4.4.3 Relative antisense activity of QDs modified peptide and ON705/PNA

SA modified QDs were coupled with peptides, ON705, and leash-PNA duplex

respectively to target nucleus of the cells and correct aberrant pre-mRNA splicing. Similar

results were obtained: 1) ON705 was bio-available in the nucleus when coupled to QDs in

the presence of LF; QDs did boost the luciferase signal ~ 2 fold in the presence of LF; 2)

None of the 5 peptides tested enhanced the nuclear uptake of the gold nanoparticles in the

absence of LF.

4.4.4 The bioavailability of antisense oligonucleotides modified by nanoparticles

A systematic study of the relationships between the availability of antisense

oligonucleotides modified with nanoparticles and chemical linkers such as protein SA and

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leash (table 4.4) and type of nanoparticles employed such as gold nanoparticle and quantum

dots (table 4.5) was performed. The concentration of ON705 was 0.3 µM in those

experimental conditions. The study showed that the availability of modified oligonucleotides

with nanoparticles was independent of the above factors (Figure 9 and 10).

Table 4.5 Chemical linkers used in the study

Sample Chemical linker used Formulation

A None ON705/PNA

B SA ON705-SA-Gold

C Thiol (-SH) ON705-S-Gold

D Leash (PNA + leash)-QD

Figure 4.9 Comparison of relative antisense activity of nanoparticles with different linkers in
the presence of LF. Formulations corresponded to the ones reported in Table 4.4.

123
 Table 4.6 Nanoparticles used in the study
Sample Nanoparticle employed Formulations

E None ON705

F Quantum dots (QD605) ON705-SA-QD

G Gold nanoparticles ON705-SA-Gold

Figure 4.10 Comparison of relative antisense activity of ON705 modified with different type
of nanoparticles in the presence of LF. Formulations corresponded to those reported in table
4.5.

124
4.5. Discussion

In this study we have employed a splice variant of the β-thalessemia intron as the

read-out for evaluating functional cellular uptake and nuclear targeting of the

oligonucleotides conjugated to a gold nanoparticle/quantum dot. The splice variant assay is

specific for quantifying the uptake of multifunctional nanoparticles into the nucleus and for

functional interaction with the spliceosome. A positive read-out in the luciferase assay

provides information on the expression of luciferase in an assay that involves tens of

thousands of cells (~ 1x 105 cell per well of 24-well plate). The results of this study indicate

that the pLuc 705 reporter gene splicing assay is an excellent method for a comparative

study, which emphasizes precision and relevance for antisense applications specific to

nuclear targeting.

The ON705-SA-gold nanoparticles were first prepared and characterized by UV-vis

spectroscopy, gel electrophoresis, DLS and fluorescence microscopy. It is worth mentioning

here that one might argue that ON705 would dissociate from gold nanoparticle surfaces once

the ON705-SA-gold vectors were incubated with LF in a cell medium, which contains

various proteins and high concentrations of salts. In order to test this, gel electrophoresis

experiment was performed on two samples: the ON705-SA-gold nanoparticles and the

incubation mixture of LF and ON705-SA-gold nanoparticles with Opti-MEM supplemented

with 10% FBS. The band shift was observed (data not shown), confirming that LF was

bound to ON705-SA-gold and did not dissociate from the nanoparticle surfaces.

Having prepared and confirmed the delivery vectors, of initial interest in this study is

whether the antisense oligonucleotides modified by gold nanoparticles are bio-available and

bio-functional at the site of the cell nucleus. To address this concern, the luciferase assay was

125
then performed with antisense oligonucleotide-gold nanoparticles in the presence of LF. The

results revealed that the antisense oligonucleotides were available to pre-mRNA and

functional in the nucleus (Figure 4.4). Further studies showed that the availability of

modified oligonucleotides with nanoparticles was independent of the kind of chemical linker

used, or the type of nanoparticles.

Moreover, it was found that the combinations of antisense oligonucleotides and

nanoparticles were more effective than free antisense oligonucleotides with no nanoparticle

modification in terms of restoring gene splicing. A nanoparticle formulation loaded with

antisense oligonucleotides and LF was up to about 5 fold more effective in increasing Luc

expression than unmodified antisense oligonucleotides with LF. The delivery efficacy was

greatly enhanced by simply attaching oligonucleotides onto gold nanoparticle surfaces. This

phenomenon occurred probably due to the fact that the local density of oligonucleotides

attached to gold nanoparticles was vastly increased compared to free oligonucleotides in the

cell media. Higher local concentrations of oligonucleotides favored the hybridization reaction

with pre-mRNA and thus corrected the aberrant splicing more efficiently.

The finding that combinations of antisense oligonucleotides and nanoparticles were

more effective than the free oligonucleotides exclude the possibility that the Luc expression

level might be completely due to excess ON705 that was not attached to nanoparticles but

rather complexed with LF. The experimental preparation procedure (the oligonucleotide-

modified gold nanoparticles were washed 3X with PBS buffer) yielded pure nanoparticle

vectors. Also, the experiments were carried out at the ON705 concentration of maximum

effective action (Figure 4.7) so that it is not possible for a minority population of free ON705

to give a signal that would compete with the population bound to nanoparticles13.

126
 In further studies, various nanoparticle formulations utilizing five peptide sequences

(Table 4.1) and two oligonucleotides: ON705 and PNA (Table 4.2) were explored with the

luciferase assay system, and no enhancement in any luciferase expression was observed

without the aid of LF for any of these formulations. The fact that the assays showed

enhanced expression of luciferase in the presence of LF demonstrates that the cause of the

lack of luciferase expression is not in the preparation or the colloidal stability of the

nanoparticles. Commercially available Lipofectamine 2000 (LF) is a proprietary cationic

lipid basically consisting of a positively charged head group and hydrocarbon chains. The

positive charge interacts with the phosphate backbone of the nucleic acid and the negatively

charged cell membrane, allowing the fusion of the liposome/nucleic acid with the cell

membrane, forming a membrane bound vesicle. After endocytosis, the complex would

escape the endosomal/lysosome pathway, diffuse through the cytoplasm and enter the

nucleus of the cell15.

All of these observations indicated the problem might have occurred during the

intracellular process (receptor binding, endocytosis, escape from endosomes/lysosome,

cytosolic trafficking or nuclear localization) of nanoparticle vectors without the presence of

LF. The nanoparticles could be endocytosed and then trapped in the endosomes/lysosome or

simply degraded by the nuclease. It is also possible that there was no effective interaction

between the particles and the cell membranes and therefore, endocytosis never occurred.

However, cellular internalization and nuclear targeting have been observed in independent

experiments1, 4. The results here suggested multiple pathways could be involved in the

process of cellular internalization and targeting16, 17. The degree to which each pathway

contributes to the overall uptake and intracellular distribution depends on a variety of factors,

127
including the structure of the peptide itself, peptide exposure, the loading density of peptide

signal on the particle surface, chemical linkage and structure of the linker among peptides,

oligonucleotides and nanoparticles, cell type, and experimental conditions.

4.6 Conclusion

Based on the work presented here, we demonstrated that the antisense

oligonucleotides modified with nanoparticles are bioavailable and biofunctional to pre-

mRNA in the nucleus of the cells. In the presence of LF, the use of the gold nanoparticles

as a platform to deliver therapeutic agents enhances the delivery efficiency compared to the

control, which consists of antisense ON705 oligonucleotides at the same concentration.

However, the targeting ability of the oligonucleotides attached to gold nanoparticles was

not improved by the conjugation of targeting peptides that facilitate endocytosis or uptake

in this particular study. These findings provide some useful cues for the proper design and

application of synthetic delivery vectors with nanoparticles.

128
4.7 References

(1) Tkachenko, A. G.; Xie, H.; Coleman, D.; Glomm, W.; Ryan, J.; Anderson, M. F.;

Franzen, S.; Feldheim, D. L. Journal of the American Chemical Society 2003, 125,

4700-4701.

(2) Zhang, F.; Andreassen, P.; Fender, P.; Geissler, E.; Hernandez, J. F.; Chroboczek, J.

Gene Therapy 1999, 6, 171-181.

(3) Feldherr, C. M.; Akin, D. Journal of Cell Science 1999, 112, 2043-2048.

(4) Tkachenko, A. G.; Xie, H.; Liu, Y. L.; Coleman, D.; Ryan, J.; Glomm, W. R.;

Shipton, M. K.; Franzen, S.; Feldheim, D. L. Bioconjugate Chemistry 2004, 15, 482-

490.

(5) Colin, M.; Maurice, M.; Trugnan, G.; Kornprobst, M.; Harbottle, R. P.; Knight, A.;

Cooper, R. G.; Miller, A. D.; Capeau, J.; Coutelle, C.; Brahimi-Horn, M. C. Gene

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