Upload
others
View
5
Download
0
Embed Size (px)
Citation preview
Targeting Transcription Factor NF-κB by Dual Functional
Oligodeoxynucleotide Complex for Inhibition of Neuroinflammation
A dissertation submitted to the
Graduate School
of the University of Cincinnati
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
in the Department of Pharmaceutical Sciences
of the College of Pharmacy
by
Jing Hu
M.S. University of Cincinnati
July 2015
Committee Chair: Jiukuan Hao, Ph.D.
ii
ABSTRACT
Because transcription factor nuclear factor (NF)-κB plays an important role in vascular
inflammation at blood-brain barrier, the present study constructed a novel DNA complex
to specifically target endothelial NF-κB for inhibition of cerebral inflammation. This
DNA complex (GS24-NFκB) contains two functional domains: a DNA Decoy for
inhibiting NF-κB activity and a DNA aptamer (GS-24), a ligand of transferrin receptor
(TfR), for targeted delivery of the DNA Decoy into brain endothelial cells. The results
indicated that GS24-NFκB was delivered into murine brain-derived endothelial (bEnd5)
cells and the delivery was specific and TfR-dependent. The DNA chimera inhibited
inflammatory responses in the cells induced by tumor necrosis factor α (TNF-α) or
oxygen-glucose deprivation/reoxygenation (OGD/R) via down-regulation of nuclear NF-
κB subunit, P65, as well as its downstream inflammatory cytokines, such as inter cellular
adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1). The
anti-inflammatory effect of the GS24-NFκB was shown as significant reduction in
monocyte adhesion to the bEnd5 cells induced by TNF-α or OGD/R after GS24-NFκB
treatment. Pharmacokinetic study suggested its poor nuclease resistance and rapid
clearance from systemic circulation. Organ uptake of GS24-NFκB including brain uptake
was much higher compared to the scramble control and high organ uptake was observed
in liver and spleen with high expression of TfR. The brain uptake is 0.39%ID/g, which is
comparable to the brain uptake of anti-TfR monoclonal antibody, OX26 (0.44%ID/g).
Intravenous injection of GS24-NFκB (15mg/kg) was able to significantly reduce the
expressions of phospho-P65 and VCAM-1 in brain endothelial cells in mouse
iii
lipopolysaccharide (LPS) induced inflammatory model, suggesting effective inhibition of
activation and transcriptional activity of NF-κB. However, the disease-modifying effects
of GS24-NFκB need to be further investigated in future. In conclusion, our approach
using DNA nanotechnology has successfully delivered the therapeutic decoy into brain
endothelial cells and induce pharmacological effect. It could be potentially applied for
inhibition of inflammation in ischemic stroke and other neuroinflammatory diseases
affecting cerebral vasculature.
iv
v
ACKNOWLEDGEMENTS
First and foremost, I would like to thank my research advisor Dr.Jiukuan Hao for his
guidance and support for my Ph.D. studies. I would like to thank him for welcoming me
into his lab five years ago when I was a master and for giving me the opportunity to
pursue a Ph.D. degree in University of Cincinnati. In Dr. Hao’s lab, I was exposed to
diverse scientific disciplines and challenging but rewarding projects. He has encouraged
me to present my work in many environments. I really appreciate his guidance and
support which has truly pushed me to reach my full potential.
I would like to acknowledge my committee members, Dr. Gary A. Gudelsky, Dr. Kevin
Li, Dr. Kim B. Seroogy and Dr. Giovanni M. Pauletti, for their valuable advice for the
project and their helpful discussions in committee meetings. In addition, I am very
grateful to Dr. Pauletti. As the director of graduate program in pharmaceutical sciences,
his help and friendship make me really enjoy the life in University of Cincinnati. I want
to express my thanks to Dr. Seroogy and Dr. Ann M. Hemmerle as our collaborators for
their help in establishing animal model and immunostaining. I also would like to thank
Dr.Gudelsky for his kindness of sharing devices with us in animal experiments.
I would like to thank Dr. Gerald Kasting and Dr. Allison Rush for helping me use their
phosphorimager. I would like to thank Dr. Peixuan Guo and Dr. Yi Shu for their help in
RNA synthesis. Many thanks to Dr.Yongguang Yang, Dr. Chaofeng Mu, Dr. He Wen,
Dr. Linli Zhou, Daniah Alwaily, Yihong Deng, Xiaoxian An, Jiraganya Bhongsatierm
and Hari Krishna Ananthula for strengthening and guiding me whenever I needed it.
vi
I would like to thank every faculty member at the University of Cincinnati, who has ever
taught and advised me. I would like to thank all the staff in college of pharmacy who has
ever helped me.
Lastly and most importantly, I would like to thank my family members: my husband Xu,
my parents and in-laws, whose support made this happen.
vii
TABLE OF CONTENTS
CHAPTER 1 INTRODUCTION ....................................................................................... 1
CHAPTER 2 REVIEW OF THE LITERATURE .............................................................. 6
2.1 Neuroinflammation ................................................................................................... 6
2.1.1 Blood-brain barrier ............................................................................................. 7
2.1.2 CECs-mediated inflammation ............................................................................ 8
2.2 NF-κB ...................................................................................................................... 10
2.3 Oligonucleotide therapy .......................................................................................... 14
2.3.1 Transcription factor decoy ................................................................................ 15
2.3.2 NF-κB decoy..................................................................................................... 16
2.4 Receptor-mediated brain delivery ........................................................................... 18
2.4.1 Transferrin receptor .......................................................................................... 19
2.5 Aptamer-mediated gene therapy ............................................................................. 21
2.5.1 Aptamers ........................................................................................................... 21
2.5.2 Aptamers in drug delivery ................................................................................ 22
2.5.3 Aptamer-therapeutic ODN conjugates ............................................................. 25
CHAPTER 3 AIMS OF THE STUDY ............................................................................. 29
CHAPTER 4 MATERIALS AND METHODS ............................................................... 30
4.1 Cell culture .............................................................................................................. 30
4.2 Animals ................................................................................................................... 30
4.3 Preparation of GS24-NFκB complex ...................................................................... 31
4.4 Cellular uptake of GS24-NFκB by bEnd5 cells ...................................................... 32
4.4.1 Flow cytometry ................................................................................................. 32
4.4.2 TfR (CD71) siRNA transfection....................................................................... 32
viii
4.4.3 Quantitative cellular uptake of 32P-labeled DNAs ........................................... 32
4.5 DNA treatments in inflammatory models in vitro .................................................. 33
4.5.1 TNF-α model .................................................................................................... 33
4.5.2 The OGD/R model............................................................................................ 33
4.5.3 Preparation of protein samples ......................................................................... 34
4.5.4 Western blot ...................................................................................................... 35
4.5.5 RT-PCR ............................................................................................................ 35
4.5.6 Adhesion assay ................................................................................................. 36
4.6 Pharmacokinetics and tissue distribution ................................................................ 37
4.7 DNA treatment in mouse LPS model ...................................................................... 38
4.7.1 DNA treatment in LPS model .......................................................................... 38
4.7.2 Brain microvessel isolation .............................................................................. 38
4.7.3 Sickness behavior analysis ............................................................................... 39
4.8 Statistical analysis ................................................................................................... 39
CHAPTER 5 RESULTS ................................................................................................... 40
5.1 Formation of GS24-NFκB complex ........................................................................ 40
5.2 Targeted delivery of GS24-NFκB into bEnd5 cells ................................................ 41
5.3 Pharmacological effect of GS24-NFκB in vitro ...................................................... 43
5.3.1 GS24-NFκB reduced nuclear P65 expression in OGD/R and TNF-α
inflammatory models ................................................................................................. 43
5.3.2 GS24-NFκB inhibited VCAM-1 expression in OGD/R and TNF-α
inflammatory models ................................................................................................. 45
5.3.3 GS24-NFκB inhibited ICAM-1 expression in TNF-α model ........................... 49
5.3.4 GS24-NFκB inhibited monocyte adhesion to bEND5 cells in TNF-α and
OGD/R inflammatory models ................................................................................... 51
5.4 Pharmacokinetics and organ distribution ................................................................ 54
ix
5.5 GS24-NFκB inhibited P65 activation and VCAM-1 expression in mouse LPS
model ............................................................................................................................. 57
CHAPTER 6 DISCUSSION AND CONCLUSION ........................................................ 62
6.1 The complexity of the role of NF-κB in CNS ......................................................... 62
6.2 Formation of GS24-NFκB conjugate ...................................................................... 65
6.3 Cellular uptake of NF-κB ........................................................................................ 66
6.4 Pharmacological effect in inflammatory models in vitro ........................................ 68
6.5 Pharmacokinetics and biodistribution ..................................................................... 71
6.6 Pharmacological effect in vivo ................................................................................ 75
6.7 Conclusion ............................................................................................................... 76
CHAPTER 7 FUTURE DIRECTIONS ............................................................................ 78
7.1 Intracellular trafficking path and subcellular localization of GS24-NFκB ............. 78
7.2 Chemical modification of GS24-NFκB................................................................... 79
7.3 Therapeutic effect in vivo ....................................................................................... 80
REFERENCES ................................................................................................................. 82
x
LIST OF FIGURES
Figure 1 Transport pathway through BBB.......................................................................... 8
Figure 2 Adhesion molecule-mediated migration of leukocytes. ..................................... 10
Figure 3 The mechanism of NF-κB activation and inhibitory effect of GS24-NFκB. ..... 12
Figure 4 TfR-mediated endocytosis .................................................................................. 20
Figure 5 The structures of (a) GS24 and (b) FB4 aptamer ............................................... 25
Figure 6 Schematic representation of noncovalent aptamer-ODN conjugate. (a) Apatmer-
streptavidin-siRNA conjugate, (b) Aptamer-sticky bridge-siRNA conjugate, (c) Aptamer-
pRNA/pRNA-siRNA conjugate........................................................................................ 27
Figure 7 Schematic representation of covalent aptamer-siRNA-PEG conjugate. ............ 28
Figure 8 Experimental time line. ...................................................................................... 39
Figure 9 Design and predicted structure of GS24-NFκB complex by M-fold. ................ 40
Figure 10 Formation of GS24-NFκB complex: the ODNs were electrophoresed in 8%
native-PAGE gel: NF-κB-decoy-1 strand (lane 1), NF-κB-decoy-2 strand (lane 2), NF-κB
decoy (lane 3), GS24-NFκB-decoy-1 (in lane 4), GS24-NFκB complex (lane 5). .......... 41
Figure 11 Flow cytometry detection: cellular uptake of GS24-NFκB complex .............. 42
Figure 12 (a) Quantitative uptake of GS24-NFκB in bEnd5 cells, Mean ± SD, n = 6-8,
**p < 0.01. (b) Representative western blotting image of TfR expression after siRNA
silencing in bEND5 cells and cellular uptake of GS24-NFκB in TfR+/- cells. Mean ± SD,
n = 3, P<0.05. .................................................................................................................... 43
Figure 13 (a) Representative image of western blotting in P65 expression in nucleus in
OGD/R model. (b) Effect of GS24-NF-κB on the nuclear level of P65 in OGD/R model,
xi
Mean ± SD, n = 3, ###P < 0.001 vs untreated control, *P< 0.05 vs corresponding scramble
controls. ............................................................................................................................. 44
Figure 14 (a) the representative image of western blotting in P65 expression in nucleus in
TNF-α model. (b) Effect of GS24-NFκB (2μM) on the nuclear level of P65 in TNF-α
model, Mean ± SD, n = 3-5, ##P < 0.01 vs untreated control, *P< 0.05 vs scramble
control, ***P< 0.001 vs NF-κB decoy treatment. ............................................................ 45
Figure 15 (a) Representative image of western blotting in VCAM-1 expression in OGD/R
model. (b) Effect of GS24-NF-κB on the VCAM-1 expression in OGD/R model, Mean ±
SD, n = 3, ###P < 0.001 vs untreated control, *P< 0.05, ***P< 0.001 vs corresponding
scramble controls. ............................................................................................................. 47
Figure 16 (a) Representative image of western blotting in VCAM-1 expression in TNF-α
model. (b) Effect of GS24-NFκB (2μM) on the VCAM-1 expression in TNF-α model,
Mean ± SD, n = 3-6, **P < 0.01 vs NF-κB decoy treatment and scramble control.(c)
Effect of GS24-NFκB (2μM) on the mRNA expression of VCAM-1 in TNF-α model,
Mean ± SD, n = 3, ##P < 0.01 vs media control, *P <0.05 vs scramble control. .............. 48
Figure 17 (a) Representative image of western blotting in ICAM-1 expression in TNF-α
model. (b) Effect of GS24-NFκB (2μM) on the ICAM-1 expression in TNF-α model,
Mean ± SD, n = 3-8, ##P < 0.01 vs media control, **P < 0.01 vs NF-κB or scramble
control. (c) Effect of GS24-NFκB (2μM) on the mRNA expression of ICAM-1 in TNF-
α model, Mean ± SD, n = 3-4, ###P < 0.001 vs media control, **P < 0.01 vs NF-κB decoy
or scramble control. .......................................................................................................... 50
Figure 18 (a) Effect of GS24-NFκB on monocytes adhesion to bEND5 cells in TNF-α
model, Mean ± SD, n = 3, ###P < 0.001 vs media control, ***P < 0.001 vs corresponding
xii
scramble controls.(b) Representative images for adhesion assay in TNF-α model (A:
media control; B:TNF-α; C: TNF-α + scramble control(4μM); D: TNF-α + GS24-NFκB
(2μM); E: TNF-α + GS24-NFκB (4μM)). ........................................................................ 52
Figure 19 (a) Effect of GS24-NFκB on monocytes adhesion to bEND5 cells in OGD/R
model, Mean ± SD, n = 3, ###P < 0.001 vs media control, ***P < 0.001 vs corresponding
scramble controls.(b) Representative images for adhesion assay in OGD/R model (A:
media control; B:OGD/R; C: OGD/R + scramble control(4μM); D: OGD/R + GS24-
NFκB (2μM); E: OGD/R + GS24-NFκB (4μM); F: OGD/R + GS24-NFκB(8μM)). ...... 53
Figure 20 Plasma concentration time profiles: (a) GS24-NFκB and TCA-precipitable
GS24-NFκB; (b) GS24-NFκB and scramble GS24-NFκB ............................................... 55
Figure 21 Organ distribution of GS24-NFκB and scramble control: (a) heart, lung,
spleen, liver, kidney and brain; (b) brain, Mean ± SD, n = 3, **P < 0.01 vs scramble
control. .............................................................................................................................. 57
Figure 22 (a) Representative image of western blotting in phospho-P65 expression in
mouse LPS model.(b) Effect of GS24-NFκB (15mg/kg) on the phospho-P65 level in
mouse LPS model, Mean ± SD, n = 5-6, ###P < 0.001 vs media control, **P < 0.01 vs
scramble control. ............................................................................................................... 59
Figure 23 (a) Representative image of western blotting in VCAM-1 expression in mouse
LPS model.(b) Effect of GS24-NFκB (15mg/kg) on the VCAM-1 level in mouse LPS
model, Mean ± SD, n = 4-5, ###P < 0.001 vs media control, *P < 0.05 vs scramble
control. .............................................................................................................................. 60
Figure 24 Effects of DNA complex on sickness behavior induced by systemic
administration of LPS, Mean ± SD, n = 4, **P < 0.01, *P < 0.05 vs baseline ................. 61
xiii
LIST OF TABLES
Table 1 Key pharmacokinetic parameters: (a) GS24-NFκB; (b) scramble GS24-NFκB . 56
Table 2 Brain uptake of GS24-NFκB verus small molecules and TfRMAb .................... 74
Table 3 Pharmacokinetic parameters for GS24-NFκB and TfRMAbs, 8D3 and R17...... 75
1
CHAPTER 1
INTRODUCTION
Central nervous system (CNS) is a complicated system which regulates and coordinates
body activities. CNS disorders lead to various problems including mobility, mental tasks
(learning and memory), basic functions (swallowing and breathing), or emotional
changes in mood. CNS diseases are a leading cause of disability and affect over 1 billion
people worldwide. Every year, over 6 million people died as a results of neurological
disorders. Approximately 25% of people between 16 and 64 years of age suffer from a
chronic neurological disability[1]. Unfortunately, these numbers are still rising. Although
various factors contribute to the diseases, including vascular disorders, injury, toxin,
infection and degeneration, one important event involved in many of these diseases is
neuroinflammation. Neuroinflammation is characterized by activation of microglia and
astrocytes, up-regulation of inflammatory mediator, disruption of the blood-brain barrier
and infiltration of peripheral leukocytes. Neuroinflammation is initiated to protect brain
from injury [2-4], but if unregulated, it may lead to secondary brain damage by itself[5].
Neuroinflammation has been implicated in various CNS diseases, including not only
acute but also neurodegenerative diseases, e.g. stroke, multiple sclerosis (MS),
Parkinson’s disease (PD) and Alzheimer’s disease[6-10].
Inflammation of blood brain barrier (BBB) plays an important role in the onset and
progression of neuroinflammation by triggering release of inflammatory mediators and
enhancing the invasion of peripheral leukocytes [11, 12]. Those inflammatory mediators
2
include CD14 LPS receptor[13], interleukin-1(IL-1)[14], TNF [15-17], cyclooxygenase-
2[18], inducible nitric oxide synthase (iNOS) [19], adhesion molecules ICAM-1 and
VCAM-1[20-22], etc. Up-regulation of the above pro-inflammatory molecules in cerebral
endothelial cells damages the barrier function of the blood-brain barrier (BBB) [23, 24]
and leads to neuronal injury and death. Compromise of BBB results in further activation
and accumulation of macrophages, microglia, leukocytes and other immune cells
throughout the CNS, which is implicated in the development of neuronal injury and
death[25, 26] . Importantly, up-regulation of proinflammatory and inflammatory
mediators is closely related to activation of transcription factor NF-κB, which is a master
switch of inflammatory gene expression [27, 28]. Activation of NF-κB induces the
expression of inflammatory genes, such as adhesion molecules (ICAM-1, VCAM-1),
iNOS and IL-1[29]. Notably, activation of NF-κB was also seen in the BBB endothelial
cells under inflammatory conditions [30-33]. Based on the central role of NF-κB in
inflammatory responses, we hypothesize that inhibition of NF-κB activity in cerebral
endothelial cells will have suppressive effects on cerebral vascular inflammation as it
targets multiple inflammatory signals instead of one inflammatory molecule. Therefore,
cerebral endothelial transcription factor NF-κB could be a new potential therapeutic
target for neuroinflammatory diseases such as ischemic stroke, MS and PD. It would be
highly significant to design therapeutic interventions that specifically target endothelial
NF-κB for restoration or modulation of the BBB functions, as this could improve the
outcome of the diseases.
In this study, NF-κB decoy serves as the therapeutic approach to inhibit transcriptional
activity of NF-κB and subsequent inflammatory responses in brain endothelial cells. NF-
3
κB decoy, a short double-stranded DNA oligonucleotide (ODN) binds to “cis”- binding
sequence (6-10bp) of NF-κB preventing cis-trans interaction between NF-κB and the
promoter region of the target genes. It has been shown that the NF-κB decoy was
effective in modulating gene expressions under experimental conditions [34-38].
Importantly, the effectiveness of these decoy ODNs against gene expression involved in
inflammatory responses in brain endothelial cells has been demonstrated by experiments
in vitro[39-41]. Thus, inhibition of NF-κB activity in the brain endothelial cells using
NF-κB decoy could be a potential therapeutic approach for neuroinflammatory disease.
However, the therapeutic use of the oligonucleotides for brain diseases is strongly
hampered by poor crossing of BBB. Most current approaches used for brain delivery of
macromolecular drugs are invasive, like intra-cerebral injection, which cause brain tissue
damage and possible infection[42]. Therefore, the development of non-invasive systemic
delivery systems is critical to eventually achieve clinical applications of the DNA decoy
approach, which can be achieved by taking advantage of receptor-mediated transport
system present on BBB. In the present study, we used a DNA aptamer (GS-24, a ligand
of TfR) as a vector to deliver NF-κB decoy into brain endothelial cells. The GS24, a
DNA aptamer (Fig. 2), can specifically bind to the extracellular domain of mouse TfR
(TfR-ECD) to be taken up by cells via TfR-mediated endocytosis. It has been
successfully used to deliver a lysosomal enzyme into deficient cells to correct defective
glycosaminoglycan degradation in the cells [43]. A novel DNA complex for brain
delivery of NF-κB decoy with a goal to inhibit cerebral vascular inflammation was
constructed, which contains two functional structures: GS24 aptamer for brain delivery
and NF-κB decoy for anti-inflammatory effect. In the study, we have evaluated: (1)
4
delivery of GS24-NFκB in vitro and in vivo; (2) anti-inflammatory effect of GS24-NFκB
under inflammatory and Oxygen Glucose deprivation/Reoxygenation (OGD/R)
conditions in vitro; (3) pharmacokinetics and organ distribution in mouse; (4) anti-
inflammatory effect in mouse inflammatory model induced by LPS.
Significance: It is well-known that a lot of efforts have been made to inhibit
neuroinflammation but new drugs against neuroinflammation has been decreasing and
results of clinical trials have been disappointing due to lack of significant therapeutic
effect, obvious harmful side effects and limitation of brain delivery. The majority of anti-
inflammatory drugs include steroid-based glucocorticoid receptor agonists and
nonsteroidal anti-inflammatory drugs (NSAIDs). Glucocorticoids is effective in the
treatment of MS via blocking the synthesis of inflammatory mediators by inhibiting NF-
κB activation [44]. However, due to the modulation of multiple non-immunological
genes, prolonged use induces a variety of side effects, such as cardiovascular disease and
osteoporosis. Moreover, the brain delivery is limited because of the export by the p-
glycoprotein (p-gp) efflux pump present on BBB [45]. NSAIDs inhibiting
cyclooxygenases (COX) are widely used against inflammation. However, they showed
lacked significant therapeutic effect in neuroinflammation [46-48] and significant CNS
side effects, including aseptic meningitis, psychosis, and cognitive dysfunction [49, 50].
There is an urgent need to develop novel treatments against neuroinflammation. Our
novel ODN-based brain drug delivery system presents several advantages. The cell-
specific and gene-specific therapeutic strategy can significantly promote therapeutic
effect and reduce harmful side effect. The DNA conjugate only targets endothelial NF-κB
and leaves neuroprotective effect of the neuronal NF-κB unaffected. The drug delivery
5
system only contains DNA, which limits immune response and rejection after repeated
long-term drug administration, making them suitable for the therapy of chronic brain
diseases[51, 52]. It can be chemically synthesized in large quantities with relatively low
cost and amenable to various chemical modifications for improved serum stability and
pharmacokinetics [52, 53]. The size of the DNA complex is relatively small compared to
large molecules, which promotes tissue penetration [52, 54]. All these features make the
gene delivery system very promising in the treatment of brain diseases. Moreover, most
previous studies on neuroinflammation focused on the microglia, astrocytes and neurons.
The present study explores the cerebral endothelial cell as a novel site of action for brain
drug delivery and neuro-vascular inflammation therapy.
Summary: The inflammation at BBB mediated by brain endothelial cells is critical to the
onset and progression of neuroinflammation. Brain endothelial cells mediated
inflammation by releasing inflammatory molecules, including cytokines, adhesion
molecules, matrix metalloproteinase and nitric oxide, which are regulated by transcription
factor NF-κB[11, 55]. In the present study, a dual functional oligonucleotide complex
was generated to inhibit NF-κB dependent inflammatory responses, which contains two
functional domains: GS24 aptamer targeting TfR on brain endothelial cells for brain
delivery and NF-κB decoy targeting endothelial NF-κB for inhibiting inflammation at
BBB. The DNA complex provides a site-specific and gene-specific strategy to inhibit
neuroinflammation with low immunogenicity, which should be an effective approach to
treat inflammation relevant brain diseases. The cellular uptake in vitro, pharmacological
effect in vivo and in vitro, and pharmacokinetics and biodistriution of the DNA complex
have been investigated in this study.
6
CHAPTER 2
REVIEW OF THE LITERATURE
2.1 Neuroinflammation
Neuroinflammation is a double-edged sword. On the one hand, it is protective by
regenerating brain tissues, isolating damaged tissues, and removing cell debris, especially
in the early stage of inflammation[56]. On the other hand, once unregulated, the
accumulation of free radicals, inflammatory molecules and leukocytes exacerbate the
inflammation and lead to further neuronal dysfunction and loss[57]. It is well-known that
brain is described as immune-privileged area due to the absence of lymphatic vessels and
presence of BBB which controls the exchange of leukocytes and inflammatory
mediators[58]. However, this immune privilege is not complete. Upon activated by
various stimuli including injury, toxin and infection, microglia produce pro-inflammatory
molecules and enhance expression of surface antigens even though they are immature
antigen-presenting cells (APCs) in normal conditions[59-62]. And antigens can be
drained into deep cervical lymph nodes by alternative routes despite of the absence of
lymphatic vessels [12, 63]. In the presence of stimuli, brain endothelial cells are also
activated and produce inflammatory molecules and free radicals and enhance the
expression of adhesion molecules, which disrupt the blood-brain barrier and allow
invasion of leukocytes and blood inflammatory molecules[11].
7
2.1.1 Blood-brain barrier
BBB is the physical, metabolic and neurological barrier that separate the CNS from the
peripheral circulation`. BBB is primarily formed by cerebral endothelial cells (CECs), as
well as astrocytes, pericytes, perivascular macrophages and basal lamina [64, 65]. The
structure of BBB is shown in Fig.1, where CECs are surrounded by a based lamina with
astrocytes opposite on their end feet. Compared to peripheral endothelial cells, CECs
have distinguished characteristics: (1) tight junction which is primarily composed of ZO-
1,occludin and claudin proteins to form the physical barrier[66]; (2) higher electrical
resistance[67]; (3) absence of fenestrae[68, 69]; (4) high density of mitochondrial[69];
(5)absence of pinocytotic vesicular transport [68, 69]; (6) P-glycoprotein-mediated efflux
pump which is associated with multidrug resistance(MDR) in tumors[70]. All these
features of CECs contribute to its barrier function of restricting the entry of blood solute
and leukocyte migration into CNS[11].
Since BBB separates brain from peripheral system to maintain the homeostasis of the
CNS, it is selectively permeable. The transport pathway through BBB is indicated in
Fig.1[71].The physicochemical properties of the substances affect their transport across
BBB, including lipid solubility, molecular weight and electrical charge. It is easier for
low molecule weight, lipophilic and un-ionized molecules to pass through BBB [72, 73].
There are also various transport and enzymatic systems present on CECs to facilitate
traverse of certain substances, such as glucose, serotonin and transferrin [74-76]. In
addition, P-glycoprotein expressed on CECs actively transports substances from the CNS
into systemic circulation to maintain the integrity of BBB[77]. Although BBB tightly
controls the transport of blood components and cellular invasion in normal condition, the
8
permeability of BBB is significantly enhanced in inflammation due to disruption of tight
junctions, or increased pinocytotic transport or formation of transendothelial channels,
resulting in further inflammatory responses and neuronal injury. Therefore, the role of
CECs at BBB in neuroinflammation cannot be ignored.
Figure 1 Transport pathway through BBB.
2.1.2 CECs-mediated inflammation
Toll-like receptors (TLRs), a type of pattern recognition receptor, recognize structurally
conserved molecules derived from pathogens and induce immune response such as
inflammation. Major histocompatibility complex II (MHCII), cell surface molecules, are
involved in presenting antigens to CD4+ T lymphocytes, activating and recruiting
cytotoxic CD4+ T cells. Both of TLRs and MHC II are usually expressed on immune
cells. However, it has been reported that their expressions were observed on CECs in
CNS inflammatory diseases like multiple sclerosis [78, 79].In addition, BBB selectively
9
allows transport of some cytokines, such as IL-1 and TNF-α, indicating the presence of
cytokine receptors on CECs, which has been verified by many studies[80-82].Therefore,
CECs can be activated by both exogenous insults and endogenous cytokines due to the
presence of antigen receptors and cytokine receptors on the cell surface.
Upon activation, CECs mediate neuroinflammation through a variety of mechanisms. (1)
In early inflammatory process, activated CECs produce important cytokines including
TNF-α, IL-1 and IL-6[83]. These cytokines exacerbate inflammation by stimulating
further release of cytokines and enhancing the permeability of BBB through
reorganization of the actin-cytoskeleton and inducing expression of adhesion
molecules[84, 85]. (2) Activated CECs promote expression of adhesion molecules,
including selecins, integrins and the immunoglobulin (Ig) superfamily[86], which are
responsible for rolling, tethering, adhesion and finally migration of leukocytes(Fig.2).
Selectins and integrins bind to their ligands on leukocytes, resulting in weak adhesion
between CECs and leukocytes [87, 88]. To achieve high affinity of binding, ICAM-1 and
VCAM-1 recognize and bind to leukocytic ligands, lymphocyte functional antigen-
1(LFA-1) and very late antigen-4(VLA-4) respectively, which account for firm adhesion
and permit the invasion of leukocytes[86, 89]. It was once supposed that leukocytes
migrated through disrupted tight junction [90]. But more evidence indicated that the
migration occurred through transcellular pathway[91]. Activation and accumulation of
leukocytes release more deleterious mediators, which lead to further endothelial injury,
opening of BBB and subsequently neuronal dysfunction and loss[92]. (3) Activated CECs
produce matrix metalloproteinases (MMPs), a family of proteolytic enzymes [93, 94].
MMPs degrade basement membrane of CECs and contribute to the opening of BBB[95].
10
(4) Activated CECs release arachidonic acid metabolism, including prostaglandins,
leukotrienes and superoxide ion, which contribute to activation of leukocytes, secretion
of cytokines and cell membrane damage of CECs[24, 96, 97]. (5) Activated CECs
produce free radical nitric oxide (NO), resulting in BBB dysfunction[98]. However, the
mechanism is not clear. In summary, CECs play an essential role in the onset and
progression of neuroinflammation.
Figure 2 Adhesion molecule-mediated migration of leukocytes.
2.2 NF-κB
Among various inflammatory mediators involved in the inflammatory responses at BBB,
NF-κB, a dimeric transcription factor, is one of the most important ones[99, 100] as up-
regulation of inflammatory molecules is closely related to the activation of NF-κB. It is
well-known that correct modulation of gene expression is essential to the normal
functions of cells and organs. Transcription factors are such kind of regulatory proteins
which regulate transcription of specific genes. Good understanding of transcription
11
factors has given an insight of pathophysiology of various diseases and offered novel
therapeutic targets.
In mammals, five different NF-κB subunits, P50, P52, c-Rel, RelA (P65) and RelB form
homo and heterodimers in various combinations[101, 102]. P50/P65 dimer is the most
abundant one and found in almost all cell types. NF-κB is expressed in almost all cell
types and a large number of genes contain the specific NF-κB binding sites in their
promoters/enhances. NF-κB regulates the expression of these genes in cell-type- and
situation-specific manner. Due to its effect on a wide range of genes, regulation of its
activity is tightly controlled at multiple levels[55]. The canonical pathway is regulated by
IκB proteins, which contain several members, IκBα, IκBβ, IκBε, P100 and P105. P100
and P105 are the precursor proteins of P52 and P50 respectively[100]. IκB proteins
preferentially bind to certain subsets of NF-κB dimers. For example, IκBα has high
binding affinity for P65/P50 dimer [103]. IκB interacts with NF-κB and masks its nuclear
translocation signal and thus sequesters NF-κB in cytoplasm[104]. But upon stimuli
including infections, stresses, free radicals and cytokines, such as IL-1 and TNF-α, IκB
proteins are phosphorylated by IKK (IκB kinase) complex, rapidly ubiquitinated and then
degraded by cytosolic proteasome[105]. IKK complex is composed of two catalytically
active kinases, IKKα and IKKβ, and the regulatory subunit IKKα (NEMO) [106, 107].
Degradation of IκB allows NF-κB free to translocate to nucleus and activate transcription
of target genes by binding to specific DNA sequences[100](Fig.3).
To obtain a maximal NF-κB response, post-translational modifications serve to regulate
NF-κB activity, including phosphorylation, ubiquitination and acetylation [108].It has
been reported that phosphprylation of P65 at Ser536 definitely contributed to the
12
transcriptional activation of NF-κB but the mechanism was not clear[55]. One possible
mechanism is that phosphorylated P65 (Ser536) facilitate the recruitment of
transcriptional coactivators [108]. It was also indicated that phosphorylation of P65
(Ser536) was involved in regulation of P65 nuclear localization[109]. The
Figure 3 The mechanism of NF-κB activation and inhibitory effect of GS24-NFκB.
phosphorylation occurred in both nucleus and cytoplasm through different kinases [110].
It seems that translational modifications are crucial in target-gene specificity and timing
of gene expressions.
In additional to activation, the termination of NF-κB is also modulated in various
manners. IκB proteins are themselves NF-κB-dependent genes, resulting in the auto-
regulatory feedback loop[111]. Generated IκB entered the nucleus and exported NF-κB to
cytosol. Translational modifications of NFκ-B subunits also contributed to termination of
13
NF-κB response through alteration of co-factor binding or degradation of NF-κB[112].
Moreover, DNA-bound P65 can be ubiquitinated and degraded by proteasome.
NF-κB is known as its crucial role in inflammation regulation through NF-κB-dependent
transcription of cytokines, chemokines, adhesion molecules and acute phase proteins [29,
113]. Activation of NF-κB is a key event in many CNS diseases such as ischemic stroke,
MS and neurodegenerative diseases [99, 113, 114], which make NF-κB a potential target
for these diseases. A lot of evidence indicated that NF-κB was activated in various cells
in cerebral ischemia, including neurons, CECs, astrocytes and microglia[99]. Although
the effect of NF-κB activation on outcome of cerebral ischemia is not determined, many
studies have proven that inhibition of NF-κB signaling did have neuroprotective effect.
Estrogen receptor and the peroxisome proliferator-activated receptor γ (PPARγ) showed
well-established neuroprotection by inhibition of NF-κB[115, 116]. In addition, a variety
of selective IKK inhibitors which inhibited nuclear translocation of NF-κB were found to
be protective in animal model of stroke [117]. NF-κB activation was also observed in MS
brain cells, including astrocytes, oligodendrocytes, microglia, and infiltrating
macrophages in or near CNS lesions [118, 119]. NF-κB-dependent genes were up-
regulated in MS brain [120, 121]. It has been demonstrated that inhibition of NF-κB was
protective in treatment of MS. Glucocorticoids (CGs) are the most widely-used and long
established immunosuppressive drugs, which have the ability to suppress transcriptional
activity of NF-κB by increasing expression of IκB and block the nuclear translocation of
NF-κB[122, 123]. In MS treatment, CGs improved disability and facilitated lesion
recovery[124]. Activation of NF-κB is also a critical event in neurodegeneration diseases,
such as PD [113]. NBD (NEMO-binding domain) peptide, which blocked the assembly
14
of IKK and thus suppressed the activation of NF-κB, had effect in reversing the
neurodegeneration in murine model of PD by protecting TH+ (tyrosine hydroxylase )
neurons from degeneration, reducing loss of dopamine production and improving
locomotor function of mice[125].
Although most of these studies focused on neurons, microglia and astrocytes, activation
of NF-κB was also reported in the BBB endothelial cells in inflammatory conditions
[126, 127] and inhibition of endothelial NF-κB activity effectively reduced expression of
adhesion molecules and monocyte adhesion to brain endothelial cells. As mentioned
above, brain endothelial cells mediated inflammation through releasing inflammatory
molecules, including cytokines, adhesion molecules and MMP, which are regulated by
NF-κB as they are NF-κB target genes. Moreover, inhibition of NF-κB reduced the
synthesis of inducible NO synthase, which subsequently decreased the release of NO.
Therefore, inhibition of endothelial NF-kB would be an attractive therapeutic strategy for
inhibiting the inflammation of BBB, blocking leukocyte migration and finally reducing
the neuronal injury.
2.3 Oligonucleotide therapy
In recent years, oligonucleotides (ODNs) as a powerful tool for modulation of gene
expression, have gained considerate attention. They provide a rational way to design
sequence-specific ligands of nucleic acids or DNA-binding regulatory proteins for
selective regulation of gene expression. The same approach may be used to design drugs
against many disease since each site of action of nucleic acids in cells presents an
opportunities for therapeutic intervention. There are three classes of therapeutic ODNs:
(1) ODN hybridizing to RNA, including antisense ODN, siRNA and ribozymes and
15
DNAzymes; (2) ODN hybridizing to DNA, including locked nucleic acid (LNA) and
peptide nucleic acid (PNA) ;( 3) ODN hybridizing to proteins or small molecules,
including aptamers and decoys[128]. The ODN therapy has been tested and developed
not only in pre-clinical experiments but also in clinical trials. The first FDA approved
antisense ODN, Fomivirsen (1999), is used to treat cytomegalovirus retinitis (CMV),
which is a synthetic ODN with phosphorothioate linkages to enhance resistance to
degradation by nucleases. Fomivirsen blocked the translation of viral mRNA by binding
to the mRNA transcribed from a key CMV gene[129].
2.3.1 Transcription factor decoy
Transcription factors are able to recognize and bind to relatively short binding sequences
even without surround genomic DNA[130]. The consensus binding site of specific
transcription factor can be developed as tools for modulation of gene expression.
Transcription factor decoys are short, double-stranded oligonucleotides, which mimic the
sequences of the binding sites in promoter/enhancer region of target genes [131]. The
decoys result in a significant reduction in or even a blockade of transcriptional activation
by interacting with transcription factors and luring them away from DNA binding sites,
which offer a powerful tool to manipulate gene expression [128, 131]. The decoy was
first described to regulate gene expression in 1990 by Bielinska el at..[34]. Decoys
specific to octamer transcription factor and NF-κB successfully inhibited their binding
activity in nucleus and octamer-dependent activation of a reporter plasmid or NF-κB-
dependent activation of the human immunodeficiency virus (HIV) enhancer. Decoy was
first used as a therapeutic approach in 1995 by Morishita el at.. The transcription factor
E2F is involved in the regulation of cell cycle-regulatory genes and the gene encoding
16
proliferating-cell nuclear antigen (PCNA). E2F decoy was employed in the treatment of
rat carotid injury, which inhibited the expression of the above genes and blocked smooth
muscle cell proliferation and neointimal hyperplasia in injured vessels[132].
In the last several decades, with a growing number of transcription factors which have
been identified, decoys have gained more and more attention to become a potential
therapy for broad clinical application. Various decoys targeting transcription factors
including NF-κB, CREB (cAMP-response element–binding protein), activator protein
1(AP-1) and signal transducers and activators of transcription-1 (STAT-1) have been
generated and the effectiveness of these decoys has been demonstrated. CREB decoy
affected CRE binding activity and inhibited cancer cell growth in vitro and in vivo. And
the inhibition appeared to be tumor-specific since the growth of normal cells were not
affected, which indicated that CREB-decoy may be developed as a potential strategy
treating cancers by regulating the expression of cAMP-sensitive genes[133]. AP-1 plays
an important role in neointimal formation after vascular injury. AP-1 decoy inhibited the
proliferation and migration of VSMCs in vitro and blocked neointimal formation after
balloon injury to the rat carotid artery [134]. STAT-1 mediates CD40 expression in
human endothelial cells, which play an important role in acute cellular rejection in small
bowel transplantation. STAT-1 decoy suppressed CD40 expression and thus improved
mucosal perfusion, and reduced graft rejection, T-cell infiltration and apoptosis in rat
small bowel allografts during acute rejection[135].
2.3.2 NF-κB decoy
NF-κB decoy is one of the most widely used decoys, which suppresses NF-κB activation
and blocks NF-κB-dependent signaling. Due to the multiple functions of NF-κB in cells,
17
NF-κB decoys have been used in a wide variety of diseases, including cardiovascular
diseases, cancer, asthma, rheumatoid arthritis (RA), skin diseases and other inflammatory
diseases[136-141]. For example, rheunatoid arthritis is known as a chronic, systemic
inflammatory disorder affecting the synovial joint. NF-κB was found to be essential to
coordinate transactivation of cytokines related to the pathogenesis. Tomita et al.
transfected synovial cells with NF-κB decoy in vitro, which significantly inhibited the
expression of inflammatory mediators, including IL-1β, IL-6, TNF-α, ICAM-1 and
MMP. Then NF-κB decoy was introduced by an intraarticular injection into rat joints in
RA model. The production of IL-1 and TNFα in the synovium of arthritic joints were
reduced and a marked suppression of joint destruction was observed [141].
NF-κB decoy has also been proven to have anti-inflammatory effect in brain disease
models. In a rat neuropathic pain model, the NF-κB decoy blocked the activation of NF-
κB and abolished the binding activity. Single endoneurial injection of NF-kB decoy, at
the site of nerve lesion, significantly alleviated thermal hyperalgesia for up to 2 weeks
and reduced the mRNA level of the inflammatory cytokines, iNOS, and adhesion
molecules at the site of nerve injury[142]. In global brain ischemia, hemagglutinating
virus of Japan–liposome complex with NF-κB decoy oligonucleotides, which was
injected through carotid artery at 1 hour after ischemia, inhibited the mRNA expression
of TNF-α, IL-1β and ICAM-1 and significantly attenuated the neuronal damage[143]. In-
vitro study has demonstrated that delivery of NF-κB decoy into brain-derived endothelial
cells (bEnd5 cell) affected NF-κB binding activity and decreased mRNA and protein
level of inflammatory molecules (ICAM-1, VCAM-1 and iNOS) as well as reduced the
adherence of bEnd5 cells to monocytes[126, 127]. Wang et al. developed a NF-κB decoy
18
loaded polylactic acid (PLA) nanoparticle, which was taken up by rat primary brain
endothelial cells and localized in cytoplasm. The released decoys retained their biologic
activity and reduced expression of tissue factor expression [144]. In summary, all the
above studies indicated that NF-κB decoy could be a promising strategy against the
neuroinflammation.
2.4 Receptor-mediated brain delivery
Although ODN therapy offers advantages over conventional drugs, the delivery of ODN
to the desired cell type, tissue or organ is the biggest obstacle to overcome, especially for
BBB which restricts the movement of drugs into the brain. To date, invasive and
noninvasive approaches have been applied to the delivery of drugs that do not have an
appreciable BBB permeability. Invasive delivery, including direct intracranial injection,
intraventricular administration and BBB disruption, causes brain tissue damage and
possible infection[42]. Therefore, the development of non-invasive systemic delivery
systems is critical to eventually achieve clinical applications of the DNA decoy approach.
Noninvasive brain delivery can be achieved by taking advantage of endogenous transport
system present at the BBB [145]. There are two main classes of transport system, carrier-
mediated transport and receptor-mediated transport (RMT). Carrier-mediated transport
systems are typically small, stereospecific pores and not applied to delivery of large
molecules such as ODN and proteins [145]. RMT system has been used for targeted
delivery of a variety of drug cargoes, such as small molecules, proteins, genes, and drug-
loaded particles [145]. The therapeutic carrier of interest can be conjugated to a ligand
targeting receptor-mediated transport system. Once binding to specific ligands, the cell
surface receptors mediated endocytosis and delivered the therapeutic agents into cells.
19
This approach requires nothing more than an intravenous injection to allow for targeted
delivery. The most important aspect of this approach is the choice of receptors and their
ligands. Various receptors on BBB have been reported for drug delivery, including
transferrin receptor (TfR) [146, 147], insulin receptor[148, 149], insulin-like growth
factor receptor[150], low density lipoprotein receptor-related protein[151] and tumor
necrosis factor[152].
2.4.1 Transferrin receptor
The transferrin receptor, a membrane glycoprotein, is involved in receptor-mediated
uptake of transferrin-bound iron, which is expressed ubiquitously in the body, including
red blood cells, erythroid cells, hepatocytes, intestinal cells, monocytes and brain
endothelial cells. Briefly, the process is initiated by the binding of Fe-transferrin to TfR,
followed by clathrin-dependent endocytosis. Fe-Tf and TfR are opsonized in clathrin-
coated vesicles and then trafficked to endosomes which mature in a unidirectional
manner from early endosomes to late endosomes and finally fuse with lysosomal
vesicles. At low PH 5.5 in endosomes, iron is released from Fe-Tf/TfR complex and
transported out of endosome. The resultant Tf/TfR complex recycles back to cell surface
and then at extracellular physiological PH7.4, Tf dissociates from TfR and goes back to
systemic circulation as well as mediates another round cycle of iron uptake. The cycle of
TfR-mediated cellular iron uptake is shown in Fig.4 [153].
20
Figure 4 TfR-mediated endocytosis
TfR is highly expressed on brain endothelial cells and the transport of Fe-Tf/TfR
complex from the blood into the cerebral endothelial cells is not different in nature from
the uptake into other cell types [154, 155]. Taking advantage of the endocytosis process
and high expression on BBB, TfR becomes a promising target for brain drug delivery. In
the beginning, Tf itself has been used as a ligand for brain delivery. Antiviral drug
azidothymidine (AZT) was encapsulated into PEGylated albumin nanoparticle (PEG-
NPs), which was modified with Tf as a ligand for brain targeting. Tf anchored PEG-NPs
significantly enhanced the uptake in brain tissue and brain localization of AZT compared
with unmodified PEG-NPs [156]. However, since TfR is almost saturated with
endogenous Tf, Tf is not an ideal ligand for brain delivery. Efforts have been made to
develop monoclonal antibody (MAb) against TfR as alternative ligands, of which binding
site is different from that of Tf. These antibodies have been widely used in brain drug
delivery in the treatment of various brain diseases, which is known as receptor-mediated
brain delivery with Molecular Trojan Horses. TfRMAb-TNFR fusion protein was
generated by fusing TNF decoy receptor to TfRMAb against mouse TfR. The fusion
21
protein was rapidly transported into mouse brain in vivo after intravenous administration
[157]. Then the fusion protein was used to treat acute brain ischemia in mouse.
Intravenous administration of the fusion protein significantly reduced hemispheric,
cortical, and subcortical stroke volumes, and neural deficit [158]. Erythropoietin (EPO),
reducing oxidative stress in brain, was fused to TfRMAb against TfR to form a brain
penetrating cTfRMAb-EPO fusion protein. Following 3 weeks of treatment with
intravenous injection of the fusion protein, PD mice showed a 306 % increase in striatal
TH enzyme activity and improvement in neuro-behavior [159]. Based on the above
previous studies, TfR, currently one of the most developed RMT-based transport system,
should be a good transport target in this study.
2.5 Aptamer-mediated gene therapy
2.5.1 Aptamers
After choosing an appropriate RMT system, the next step is to find targeting ligand.
Ligands generally contain antibodies, peptides, small molecules, and aptamers[160]. In
this study, we used aptamer targeting TfR to delivery NF-κB decoy into brain endothelial
cells. Aptamer are oligonucleotides selected in vitro from complex nucleic acid libraries
by a process called SELEX (systematic evolution of ligands by exponential enrichment)
to bind target molecules with high affinity and specificity because of their stable three-
dimensional structure [52]. It has been more than 20 years since aptamer was first
described. Over the last decades, aptamers have been successfully developed as
therapeutic or diagnostic approaches. For example, Pegaptanib (brand name Macugen) is
a pegylated anti-vascular endothelial growth factor (VEGF) aptamer, which was
approved by FDA in 2004 to treat neovascular (wet) age-related macular degeneration
22
[161]. Due to its ability to bind to cell surface receptors with high affinity and specificity
and subsequently induce cellular endocytosis, the application of aptamer in drug delivery
has attacked more and more interests.
Compared to other ligands, aptamer have several advantages. (1) Aptamer has much
higher targeting selectivity than small molecules such as folic acids although they have a
low molecular weight and good stability [162, 163]. (2) Peptides are unstable and
degraded easily in systemic circulation while aptamer is amenable to chemical
modification to improve its serum stability and pharmacokinetics [164, 165]. (3)
Although both aptamer and antibody show high affinity and specificity to targets,
aptamers have better tissues penetration due to lower molecular weight [52, 166]. (4)
Antibody often induce strong immune responses, especially following repeat injections
while aptamer has limited immune response and rejection after repeated long-term drug
administration [166, 167]. (5) Aptamer is more thermal stable than antibody. Aptamer is
able to regain its 3D conformation once cooled to the room temperature after
denaturation at 95 °C while antibody is permanently inactivated at high temperature
[166]. (6) Compared to antibody, aptamer can be chemically synthesized in large
quantities with lower cost. All these features make aptamer a popular ligand for targeted
drug delivery.
2.5.2 Aptamers in drug delivery
Many of aptamers have been selected, identified and used in targeted drug delivery for
the treatment of various diseases. These aptamers target a variety of cell surface
receptors, including prostate-specific membrane antigen (PSMA)[168-173], nucleolin
[174, 175], mucin-1(MUC1) [176], epidermal growth factor receptor (EGFR) [177, 178],
23
HIV protein gp120 [179, 180], and TfR [43, 181]. PSMA aptamer A10 and its truncated
secondary generation A10-3.2, conjugated with siRNAs targeting prostate cancer–
specific pro-survival genes, successfully targeted PSMA-expressing tumors and inhibited
tumor growth in vitro and in vivo [168, 169]. Boyacioglu et al. identified a new DNA
aptamer and developed dimeric aptamer complexes (DACs) for specific delivery into
PSMA-positive tumor cells [171]. Nucleolin aptamer AS1411 has been demonstrated to
not only have anti-tumor effect itself but also facilitate tumor-specific delivery of cargoes
to enhance their anti-tumor effect and reduce side effect [174, 175, 182]. Mucin-1 and
EGFR aptamers also mediated tumor-targeted delivery [176, 177]. Aptamer (UCLA1)
targeting gp120 expressed on human immunodeficiency virus-1(HIV-1), has been
employed to deliver anti-human immunodeficiency virus (anti-HIV) siRNAs into HIV-
infected cells [179].
Aptamers targeting TfR: Chen et al. first selected and isolated a DNA aptamer (GS24)
(Fig.5a) and a RNA aptamer (FB4) (Fig.5b) targeting mouse TfR. To investigate its role
in mediating endocytosis, the two aptamers were modified with biotin, followed by
linked to dye-labeled streptavidin for detection by confocal microscopy. The aptamer-
streptavidin conjugates have been successfully internalized into mouse fibroblasts (Ltk−
cells) and the internalizations appeared to be specific since they were inhibited by an
excess of free aptamers. Moreover, the uptake was mouse TfR-dependent because the
conjugates were not taken up by human cells (293T) unless they were firstly transfected
with mouse TfR. The fact that the internalization was not inhibited by transferrin at a
concentration similar to that found in mouse serum indicated that the two aptamers bind
to extracellular domain of the mouse TfR which was different from the binding site of
24
transferrin, therefore avoiding competition for binding site with Tf and limiting effects on
normal functions of TfR. In addition, aptamer-streptavidin followed a different cellular
trafficking path from transferrin. After 30min of endocytosis, transferrin was not
observed in cells since it released the iron and recycled back to cell membrane while the
conjugates colocalized with dextran-labeled lysosomes. They indicated that the
endocytosis of the conjugates were still mediated by coated pits because it was not
inhibited by methyl-β-cyclodextrin, an agent inhibiting alternative endocytic pathway by
lipid rafts and caveolae. In the treatment of lysosomal storage diseases, GS24 was
conjugated with a lysosomal enzyme, which successfully delivered the enzyme into
lysosomes and relieved the enzyme deficiency [43]. In our previous study, ICAM-1
siRNA was internalized into brain-derived endothelial cells (bEnd5 cells) via RNA
aptamer FB4-mediated endocytosis and the siRNA effectively blocked the expression of
target gene [181]. Because of high expression of TfR on brain endothelial cells, TfR
aptamer have become a promising brain delivery approach to overcome the challenge of
blood-brain barrier (BBB). In summary, it is very important to choose the perfect target
for aptamer-mediated drug delivery to achieve high target specificity, which should be
highly expressed on the target cells but not or lowly expressed on the surface of nontarget
cells.
25
(a)
(b)
Figure 5 The structures of (a) GS24 and (b) FB4 aptamer
2.5.3 Aptamer-therapeutic ODN conjugates
Aptamer has been widely used in targeted delivery of ODN-based therapeutic molecules,
including siRNA, miRNA, shRNA and decoy ODN. Aptamer can be connected with
therapeutic ODNs noncovalently or covalently.
In noncovalent conjugates, connectors have been employed, including streptavidin, GC-
rich “sticky sequence”, and packaging RNA. Taking advantage of high binding affinity of
streptavidin for biotin, Chu et al. generated an aptamer:streptavidin:siRNA conjugate, in
which two biotinylated-PSMA aptamer A9 were connected to two biotinylated siRNA
via one molecule of streptavidin as a connector(Fig.6a). The conjugated was proven to be
taken up by PSMA-positive cells and lead to gene silencing in these cells [168]. Zhou et
al. developed a gp120 aptamer-anti-HIV siRNA conjugate with a sticky bridge. 3′-end of
the aptamer and one of the two siRNA strands were chemically synthesized with a 3′ 7-
26
carbon linker (7C3), which in turn was attached to a 16-nucleotide 2′ OMe/2′ Fl GCrich
complementary bridge sequences. Then the aptamer-siRNA noncovalent conjugate can
be formed via Watson-Crick base-pairing by simple mixing (Fig.6b). siRNA was
demonstrated to be released from the conjugate and trigger RNAi. Systemic
administration of the conjugates inhibited HIV-1 viral load in viremic RAG-hu mice and
did not induce any innate inflammatory cytokine production [180]. In our previous study,
packging RNAs (pRNAs), derived from bacteriophage phi29 was used as a vector to link
ICAM-1 siRNA and TfR aptamer (FB4) as a conjugate (Fig.6c). pRNAs contain two
functional domains: the double-stranded helical domain at 5’/3’ end and the
intermolecular binding domain . These two domains fold independently, and change of
the primary sequences of helical region doesn’t affect pRNA structure and folding as
long as the two strands are paired. Therefore, the helical region at the 5’/3’ end of pRNA
can be replaced by siRNA or aptamer without affecting the formation of RNA multimers
mediated by base-pairing of upper and lower loops in the intermolecular binding domain.
Our siRNA-pRNA-aptamer chimera have been specifically delivered into TfR-positive
bEnd5 cells but not TfR-silenced cells. siRNA was released from chimera by dicer
cleavage, which successfully blocked the expression of ICAM-1[181].
27
(a)
(b)
(c)
Figure 6 Schematic representation of noncovalent aptamer-ODN conjugate. (a) Apatmer-
streptavidin-siRNA conjugate, (b) Aptamer-sticky bridge-siRNA conjugate, (c) Aptamer-
pRNA/pRNA-siRNA conjugate.
Aptamer-ODN covalent conjugates only involve nucleic acids, which largely reduces
immunogenicity, making it suitable for in vivo application. McNamara et al. first
generated a completely RNA-based A10-siRNA chimera, which targeted PSMA and
28
delivered therapeutic siRNAs against two survival genes PLK1 (polo-like kinase 1) and
BCL2 (B-cell lymphoma-2) to tumor cells. The DNA encoding A10-siRNA was prepared
and then A10-siRNA conjugate was obtained by transcription, in which 2’-fluoro
modified A10 was covalently linked to the sense strand of siRNA and then annealed with
the guide strand. The chimera was specifically taken up by PSMA expressing cells and
siRNA was released by dicer to suppress the expression of target genes and induce cell
death. In a xenograft model of prostate cancer, intratumoral delivery of the chimera
displayed anti-tumor effect [53]. Then the chimera was optimized by Dassie et al. to
obtain anti-tumor activity following systemic administration. A10 was replaced by its
truncated version A10-3.2 for easier chemical synthesis. 2-nt (UU)-overhang was
introduced at the 3’ end of the siRNA duplex for better recognition by dicer and
passenger and guide strands of the siRNA were swapped to facilitate 5’ terminal
modifications of the shorter RNA strand without loss of function. To enhance the
circulating half-life, a 20 kDa PEG was appended to the 5’-terminus of the smaller RNA
strand without affecting chimera targeting and silencing (Fig.7) [169].
Figure 7 Schematic representation of covalent aptamer-siRNA-PEG conjugate.
29
CHAPTER 3
AIMS OF THE STUDY
The overall objective of the study was to develop a novel gene brain delivery system to
inhibit neuroinflammation by blocking the activation of inflammatory mediator, NF-κB.
This work has potential applicability to the development of therapies for
neuroinflammation relevant CNS diseases. We hypothesize that the DNA complex
(GS24-NFκB) containing GS24 aptamer for brain delivery and NF-κB decoy for blocking
NF-κB activation, is able deliver NF-κB decoy into brain endothelial cells via TfR-
mediated endocytosis and the delivered decoy is able to inhibit transcriptional activity of
NF-κB and NF-κB dependent inflammation.
1, The first aim was to evaluate the cell uptake of GS24-NFκB. The capability and
specificity of the cellular uptake were studied.
2, The second aim was to identify the pharmacological effect of GS24-NFκB in two in
vitro inflammatory models: TNF-α model and OGD/R model. The activation and
transcriptional activity of NF-κB were investigated.
3, Under the third aim, the pharmacokinetics and organ distribution of GS24-NFκB were
defined following systemic administration.
4, Our fourth aim was to identify the pharmacological effect of GS24-NFκB in mouse
LPS-induced inflammatory model. The activation and transcriptional activity of NF-κB
in isolated brain microvessels were measured and neurological deficiency were studied.
30
CHAPTER 4
MATERIALS AND METHODS
4.1 Cell culture
The brain endothelial cell line (bEnd5) derived from mouse brain and immortalized with
polyoma middle T oncogene was gifted by Dr. Ulrich Bickel, Texas Tech University.
bEND5 cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Hyclone,
Logan, UT, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine
serum(FBS) (Atlanta Bio InC. GA, USA), 1% (v/v) non-essential amino acids and 1%
(v/v) 10000IU/ml penicillin/ 10000µg/ml streptomycin (all from ATCC, Manassas, VA,
USA). U937 cells (a monocyte cell line from ATCC, Manassas, VA, USA) were cultured
in RPMI 1640 medium (Hyclone, Logan, UT, USA) supplemented with 2 mM L-
glutamine (Mediatech Inc. Cellgro, Manassas, VA, USA), 10% heat-inactivated FBS and
1% (v/v) 10000IU/ml penicillin/ 10000µg/ml streptomycin. Both cell lines were
maintained at 37 °C, 5% CO2 and 95% relative humidity.
4.2 Animals
All animal procedures were approved by the Institutional Animal Care and Use
Committee at University of Cincinnati and they complied with pertinent NIH guidelines
for care and use of animals. CD1 Male mice (body weight ~25g) supplied by Charles
River (Wilmington, MA, USA) were kept under standardized light/dark (12 hours),
temperature (22°C), and humidity (70%) conditions, with free access to water and
standard pelleted food.
31
4.3 Preparation of GS24-NFκB complex
The sequence of GS24-NFκB is:
GS24-NFκB-decoy-1 strand:5’- GCG TGT GCA CAC GGT CAC TTA GTA TCG CTA
CGT TCT TTG GTT CCG TTC GGC CTT GAA GGG ATT TCC CTC C -3’
NF-κB decoy-2 strand: 5’-GGAGGGAAATCCCTTCAAGG-3’.
The sequence of scramble GS24-NFκB is:
Scramble GS24-NFκB -decoy-1 strand:5’- AAG AGA GTA AAT CCT GGG ATC ATT
CAG TAG TAA CCA CAA ACT TAC GCT GGC CTT GAA GGG ATT TCC CTC C-
3’.
NF-κB decoy-2 strand: 5’-GGAGGGAAATCCCTTCAAGG-3’
The sequence of NFκB-decoy is[35]:
NFκB-decoy -1 strand: 5’-CCT TGAAGGGATTTCCCTCC-3’.
NF-κB decoy-2 strand: 5’-GGAGGGAAATCCCTTCAAGG-3’
The designed sequences of the strands above were purchased from Integrated DNA
Technologies Inc (Coralville, Iowa, USA). The ODN complex was constructed by DNA
denaturation and annealing process. Briefly, the mixture of two complementary strands
(mole ratio 1:1) in annealing buffer (10mM Tris, PH 7.5-8.0, 50mM NaCl, 1mM EDTA)
was incubated at 95°C for 5 min, and then cooled at the room temperature for 45-60 min.
The DNAs were analyzed by 8% native-PAGE gel.
32
4.4 Cellular uptake of GS24-NFκB by bEnd5 cells
4.4.1 Flow cytometry
GS24-NFκB and naked NF-κB decoy were labeled with Cy3 using the Mirus Label IT
Cy3 labeling kit (Mirus bio LLC., Madison, WI, USA) according to the manufacturer’s
instructions. BEnd5 cells were seeded in 12-well plates 24 hours before adding Cy3
labeled DNAs. Cells were incubated with DNAs for 30mins at 37°C and then washed,
trypsinized, pelleted and suspended in FACS Buffer (PBS, 0.5% BSA and 2mM
EDTA)[183]. The cell suspension was kept on ice and analyzed on a FACSCaliburTM
flow cytometer (Becton Dickinson, Mountain View, CA).
4.4.2 TfR (CD71) siRNA transfection
The TfR negative bEnd5 cells were generated by transfecting CD71 siRNA with
lipofectamine RNAiMAX (Life Technology, Grand Island, NY) according to
manufacturer’s protocol.
4.4.3 Quantitative cellular uptake of 32P-labeled DNAs
GS24-NFκB and its scramble control were 5’-end labeled with [γ-32P] ATP (PerkinElmer
Inc.,Waltham, Massachusetts, USA) using T4 Polynucleotide Kinase (Fermentas, USA)
according to the manufacturer’s instructions, and then purified by NucAway™ spin
column (life technology, Grand Island,NY). The purity of the product was controlled by
precipitation of the ODNs with trichloroacetic acid (TCA) (Sigma-Aldrich Co.LLC., St.
Louis, MO, USA), and only the batches with precipitation >95% were used [184].
Briefly, ice-cold 10% TCA (500µl) was added to a mixture of 1 μl sample and 50µl 2.5%
bovine serum albumin (BSA, Fisher Scientific, Pittsburgh, PA, USA). The resulting
mixture was then vortexed and incubated on ice for 10 min following by centrifugation at
33
4000g for 5 min. The supernatant was collected and the pellet was dissolved in 3% KOH.
The radioactivity of TCA-precipitable fraction was calculated as % (precipitable fraction)
= (CPMpellet X 100)/ (CPMpellet +CPMsupernatant).
The TfR negative and normal bEnd5 cells with 80-90% confluence were incubated with
32P-labeled DNAs at the concentration of 1µmol/l (~0.4 µCi) for 40 minutes at 37 °C.
After washed three times with PBS, the cells were lysed with RIPA buffer and the cell-
associated radioactivity was counted by LSC 6500-multiplepurpose scintillation counter
(Beckman Counter, Fullerton, CA). For competition assay, 20-fold extra unlabeled GS24-
NFκB was added to the medium 30 min before adding 32P-labeled DNAs.
4.5 DNA treatments in inflammatory models in vitro
4.5.1 TNF-α model
Briefly, the confluent bEnd5 cells were firstly incubated with GS24-NFκB or its
corresponding controls in serum free medium for 2 hour. Then TNF-α (50 µg/ml)
(Shenandoah Biotechnology, Warwick, PA, USA) was added to the medium to stimulate
inflammation of bEen5 cells for 4 hours, and then cell samples were harvested.
4.5.2 The OGD/R model
The OGD/R model was used to mimic ischemia/reperfusion in vitro. Briefly, hypoxia
was induced by placing cells in a sealed chamber (BillupsRothenberg, Del Mar, CA) at
37 °C, which has been flushed with 95% N2 /5% CO2 gas. The concentration of oxygen
in the atmosphere was maintained at 0% oxygen and the PO2 in the medium was below
25 mmHg. Aglycemia was induced by using RPMI 1640 medium (without L-glucose)
(Hyclone, Logan, UT). After 9 hours OGD, the cells were back to normal growth
condition for 16 hours. Then the growth medium was changed to serum free medium and
34
the cells were incubated with GS24-NFκB or its corresponding controls for another 8
hours. The cell samples were obtained at 24 h after reoxygenation.
4.5.3 Preparation of protein samples
4.5.3.1 Whole cell lysate
Cells were sonicated briefly in ice-cold 1X RIPA buffer containing PMSF, sodium
orthovanadate and protease inhibitor cocktail solution (Santa Cruz Biotechnology, Inc.
CA). After incubation in ice-cold buffer for 30 minutes, centrifuge tissue/cell lysate at
10,000xg for 10 minutes at 4°C. The supernatant fluid was harvested for the following
steps. An appropriate volume of Laemmli sample buffer with 2-mercaptoethanol was
added to the homogenate, and the samples were incubated in a water bath at 100°C for 8
min[185].
4.5.3.2 Nuclear protein extraction
Cells were washed and lysed in ice cold buffer-A containing 10 mM HEPES pH 7.9, 1.5
mM MgCl2, 10mM KCl, 0.25% v/v noident P-40, 0.5 mM dithiothreitol (DTT), 0.5 mM
phenylmethylsulfonyl fluoride (PMSF) in deionised water (dH2O) for 10 min. The
supernatant cytoplasmic and nuclear pellet fractions were obtained by centrifuging cell
samples at 12,000g for 2 min. Nuclear protein was extracted from the pellet with the ice
cold buffer-B containing 20 mM HEPES pH 7.9, 1.5 mM MgCl2, 0.42 M NaCl, 0.5 mM
DTT, 25% v/v Glycerol, 0.5 mM PMSF in diH2O for 20 min. Then the nuclear fractions
were collected and diluted in the buffer-C containing 20 mM HEPES pH 7.9, 50 mM
KCl, 0.5 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF[186]. Finally, the protein
35
concentrations were measured, and western blotting was performed. All the chemicals
were purchased from Sigma-Aldrich Inc.
4.5.4 Western blot
Protein samples were loaded onto SDS-polyacrylamide gels (Bio-Rad, Hercules, CA),
and ran for 1.5 h at 120 v. The proteins were transferred with a semidry transfer cell (Bio-
Rad) over night at 4°C. After transfer, the membranes were blocked with 5% fat-free
milk in 10 mM Tris, 100 mM NaCl, and 0.1% Tween 20 (TBST; pH 7.5) for 1 h. The
membranes were washed three times for 15 min with TBST and incubated with
corresponding primary antibodies overnight at 4°C, followed by three additional washes
with TBST for 15 min and incubation with secondary antibody for 1 h. Final detection
was carried out with enhance chemiluminescence methodology (Pierce Supersignal West
Dura) and the intensity of the signal was measured with software AlphaEaseFC. Nuclear
expression of P-65 and expression of ICAM-1 and VCAM-1 were determined. The β-
actin was used as the protein loading control marker for whole cell lysate and TATA
binding protein (TBP) was used as protein loading control marker for nuclear protein.
The following antibodies and the respective dilutions were used in western blotting
procedure: 1: 2,000 (dilution) β-Actin antibody (Cell Signaling Technology, Danvers,
MA), 1:1,000 ICAM-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), 1:2,000
VCAM-1 antibody and 1:1,000 TBP antibody (Abcam, Cambridge, MA)[185].
4.5.5 RT-PCR
Total RNA was extracted using the ready-to-use reagent TRIzol (life technology).
Briefly, the medium was removed and TRIzol was added to each well and the cells were
lysed by pipetting the cells up and down several times. After incubation at room
36
temperature for 5 min, the homogenized samples were well mixed with chloroform and
incubated for another 2 min. Then the samples were centrifuged at 12,000 × g for 15
minutes at 4°C. The aqueous phase was transferred into a new tube and then 100%
isopropanol was added into the tube. After 10 min incubation on ice, the samples were
centrifuged at 12,000 × g for 10 minutes at 4°C. The RNA pellets were washed with 75%
ethanol and air dried. Nuclease-free water was used to prepare RNA samples for further
analysis. Then cDNAs were obtained using the RNA as template and high-capacity
cDNA reverse transcription Kit (life technology) according to manufacturer’s protocol.
Finally, RNA were quantified by quantitative real-time PCR with two gene specific
primers and SYBR® Green real-time PCR master mixes (life technology). The following
forward and reverse primers were used[126]: VCAM-1: 5'-AAG ACT GAA GTT GGC
TCA CAA-3' and 5'-GGA GTT CGG GCG AAA AAT AG-3'; ICAM-1: 5'-GGG AAT
GTC ACC AGG AAT GT-3' and 5'-CAG TAC TGG CAC CAG AAT GA-3' ; β-actin:
5'-GGC TGT ATT CCC CTC CAT CG-3' and 5'-CCA GTT GGT AAC AAT GCC ATG
T-3'. The mRNA expression of β-actin was used as internal control. All the primers were
purchased from Integrated DNA Technologies Inc.
4.5.6 Adhesion assay
U937 cells (a monocyte cell line from ATCC) were incubated with 5 mg/mL BCECF-
AM (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester, Sigma Inc.)
for 30 minutes at 37°C, and then the cells were washed and resuspended in serum free
media. For ODN treatments in TNF-α stimulated bEND5 cells, bEND5 cells were
cultured and incubated with ODNs in a 24-well plate for 2 h. Then TNF-α (20ng/ml) was
added and incubated for 16 h, and then the cells were incubated with BCECF-AM-
37
labeled U937 cells (106cells/well) for 30 minutes at 37°C. For ODN treatments in
OGD/R condition, the bEND5 cells were treated as described early in OGD/R model, and
then cells were incubated with BCECF-AM-labeled U937 cells (106cells/well) for 30
minutes at 37°C. Non-adhering U937 cells were removed and cells were washed with
PBS, then cells were lysed in 0.1% Triton X-100 in 0.1M Tris-HCl (pH7.4) (Sigma-
Aldrich). Fluorescence (F) was measured with a microplate fluorescence reader (POLAR
star OPTIMA, BMG Labtechnologies, Ortenberg, Germany) using excitation at 492 nm
and emission at 535 nm. The monocyte adhesion was calculated as: Adhesion (%)
=100×Fsample/F total (F: fluorescence intensity of 106cells) [126].
4.6 Pharmacokinetics and tissue distribution
Male CD1 mice (body weight ~25g, supplied by Charles River Inc, Wilmington, MA,
USA) were anaesthetized by injection of Ketamine and Xylazine (Butler Animal Health
Supply Inc. Dublin, OH, USA). 32P-labeled ODNs (13µg, 100μl, ~2μCi) were
intravenously administered through jugular vein. Blood samples were obtained by orbital
puncture at 0, 2, 5, 10, 20, 30, 45 and 60 min. And then mice were sacrificed by
decapitation and organs including lung, heart, spleen, kidney, liver and brain were
sampled and weighed. Blood was centrifuged at 2000g and 4°C for 10min, and the
plasma was collected[187]. The radioactivity of the plasma was measured. One aliquot of
plasma was treated with TCA precipitation to obtain the TCA-precipitable radioactivity
[184]. The radioactivity levels in the injected solutions serve as internal standards. The
plasma concentrations were expressed as percentage of injected dose per µl of plasma at
the various time points. Concentration-time data were fitted to bioexponential equation
and analyzed by PKSolver. The organs were cut into pieces and solubilized in 3% KOH
38
at 50°C for 4 h as well as decolorized in 5% H2O2. Radioactivity of all samples was
measured by LSC 6500-multiplepurpose scintillation counter. DNA accumulations in the
organs were expressed as percent of injected dose %ID/g. The results were corrected for
capillary plasma content in organs, 48.2, 170, 140, 114, 83, 9.3 μl/g for heart, lung, liver,
spleen, kidney and brain respectively.
4.7 DNA treatment in mouse LPS model
4.7.1 DNA treatment in LPS model
Lipopolysaccharide (LPS, Sigma-Aldrich) was dissolved in sterile saline, aliquoted, and
stored at -20°C until use. LPS model was established by a single intraperitoneal (i.p.)
injection with 1mg/kg LPS. For measurement of phospho-P65 expression, at 3 h after
LPS challenge, GS24-NF-κB or its scramble control was administered by intravenous
(i.v.) injection via jugular vein with the dose of 15mg/kg. Brain samples were obtained
after 3 h of DNA treatment. Phospho-P65 (Ser536) antibody was purchased from Cell
Signaling Technology. For analysis of VCAM-1 expression, at 16 h after LPS injection,
GS24-NFκB or its scramble control was administered by i.v. injection with the dose of
15mg/kg. Brain samples were collected after 4 h of DNA treatment (Fig.8).
4.7.2 Brain microvessel isolation
Brain samples were homogenized in a tissue grinder with ice cold medium 131 (life
technologies, Grand Island, NY) supplemented with 2% FBS, 1% (v/v) 10,000 IU/ml
penicillin/10,000 mg/ml streptomycin. The homogenates were suspended in 16% dextran
(average molecular weight 100,000g, Sigma-Aldrich) and centrifuged at 5000RPM for 20
min. Then the supernatant containing fatty layer was removed and vascular pellet was
digested with 0.1% collagenase and 10µg/ml DNase at 37°C for 1 h with occasional
39
agitation. The digested microvessel was filtered through a 70µm nylon mesh cell filter
and the microvessels were collected with centrifugation at 1000g for 5 min [188]. Then
the cells were lysed and used for further analysis.
4.7.3 Sickness behavior analysis
The mouse was placed individually in a new cage similar in all aspects to the homecage
and videotaped for 5 min and then moved back to homecage. The recordings were
analyzed offline by overlapping a grid consisting of 12 identical rectangles (3-by-4 array)
on the cage floor and counting the number of rectangles the mouse has entered with all
four paws during the test period. The spontaneous locomotor activity of each mouse was
tested before LPS injection, at 16 h after LPS injection and at 4 h after DNA treatment
(Fig.8a). The activity measured before LPS injection was set as baseline and the activity
following the LPS challenge was expressed as percentage of baseline activity[189].
(a) (b)
Figure 8 Experimental time line.
4.8 Statistical analysis
Results are expressed as the mean ± S.D. of n experiments. Statistical analysis was
performed by analysis of variance, followed by posttests (Dunett’s test for comparison of
experimental groups versus control conditions). A p value of < 0.05 was chosen as the
threshold of significance.
40
CHAPTER 5
RESULTS
5.1 Formation of GS24-NFκB complex
The design and sequence of GS24-NFκB is shown in Fig.9: NF-κB 2 sequence is
complementary to the NF-κB1 sequence in the 3’ end of GS24-NFκB1 (showed in bold)
to form GS24-NFκB DNA. The electrophoretic gel Image (Fig.10) indicated that the
oligonucleotides with different molecular weight exhibited different migration rates. The
migration distance of GS24-NFκB is the shortest due to its biggest molecular weight,
which verified the formation of GS24-NFκB complex.
Figure 9 Design and predicted structure of GS24-NFκB complex by M-fold.
41
Figure 10 Formation of GS24-NFκB complex: the ODNs were electrophoresed in 8%
native-PAGE gel: NF-κB-decoy-1 strand (lane 1), NF-κB-decoy-2 strand (lane 2), NF-κB
decoy (lane 3), GS24-NFκB-decoy-1 (in lane 4), GS24-NFκB complex (lane 5).
5.2 Targeted delivery of GS24-NFκB into bEnd5 cells
To evaluate the cellular delivery capability of aptamer GS24, the DNA complex and
naked NF-κB decoy were labeled with Cy3. Cellular binding and internalization were
indicated by cell-associated fluorescence signals, which were observed and analyzed by
FACS. The results in Fig.11, suggested a significant shift for GS24-NF-κB complex but
no shift for naked NF-κB decoy, which indicated that GS24-NF-κB showed higher
cellular binding/uptake efficacy but NF-κB decoy without aptamer showed no
binding/uptake.
We further investigated the quantitative uptake of the DNA complex and the delivery
specificity. DNAs were 5’-end labeled with [γ-32P] ATP and cell-associated radioactivity
represented the cellular binding and internalization. The radioactivity for GS24-NFκB
treatment was 42491±11668 (mean±SD) CPM while it was 6220±3621 CPM for
scramble control, which suggested that cellular uptake of GS24-NF-κB was much higher
42
(5.8 fold) than that of scramble control. The results of competition assay indicated that in
the presence of extra unlabeled DNA complex, the delivery of GS24-NFκB into bEnd5
cells was reduced by 90% with radioactivity of 388±49 CPM (Fig.12a). Anti-TfR siRNA
was transfected into bEnd5 to block the expression of TfR, which was verified by
western blot (Fig.12b). The uptake of GS24-NF-κB in normal bEnd5 cells was 3.6±0.7
times of that in the TfR silencing cells (Fig.12b).
Figure 11 Flow cytometry detection: cellular uptake of GS24-NFκB complex
43
(a) (b)
Figure 12 (a) Quantitative uptake of GS24-NFκB in bEnd5 cells, Mean ± SD, n = 6-8,
**p < 0.01. (b) Representative western blotting image of TfR expression after siRNA
silencing in bEND5 cells and cellular uptake of GS24-NFκB in TfR+/- cells. Mean ± SD,
n = 3, P<0.05.
5.3 Pharmacological effect of GS24-NFκB in vitro
5.3.1 GS24-NFκB reduced nuclear P65 expression in OGD/R and TNF-α
inflammatory models
P65 is an important subunit of NF-κB. bEnd5 cells stimulated by TNF-α or subject to
OGD/R were treated with DNAs and then nuclear proteins were extracted and nuclear
P65 levels were measured. In OGD/R model, nuclear P65 level was increased to
1.95±0.13 times of the untreated control after OGD/R. But the increase was obviously
reversed by DNA treatments in a dose dependent manner. The nuclear P65 levels were
136± 20, 136± 14, and 99 ± 20% of untreated control in GS24-NFκB treatment groups at
concentrations of 2, 4, and 8 µM respectively. The scramble control did not significantly
44
affect the nuclear P65 level, which were 184±26, 182±14, and 165±23% of untreated
control at concentrations of 2, 4, and 8 µM respectively (Fig.13). TNF-α, as an
inflammation factor, also enhanced expression of nuclear P65, which was 204±38% of
control after TNF-α stimulation. But the levels were reduced to 133±21% with DNA
treatment at a concentration of 2 µM. At the same concentration, naked NF-κB decoy and
scramble control had no significant effect on the nuclear P65 levels, which were 215±52
and 181±26% relative to untreated control (Fig.14).
(a)
(b)
Figure 13 (a) Representative image of western blotting in P65 expression in nucleus in
OGD/R model. (b) Effect of GS24-NF-κB on the nuclear level of P65 in OGD/R model,
Mean ± SD, n = 3, ###P < 0.001 vs untreated control, *P< 0.05 vs corresponding scramble
controls.
45
(a)
(b)
Figure 14 (a) the representative image of western blotting in P65 expression in nucleus in
TNF-α model. (b) Effect of GS24-NFκB (2μM) on the nuclear level of P65 in TNF-α
model, Mean ± SD, n = 3-5, ##P < 0.01 vs untreated control, *P< 0.05 vs scramble
control, ***P< 0.001 vs NF-κB decoy treatment.
5.3.2 GS24-NFκB inhibited VCAM-1 expression in OGD/R and TNF-α
inflammatory models
We investigated the expression of NF-κB-dependent inflammatory gene VCAM-1. Under
OGD/R condition, VCAM-1 expression was significantly enhanced, which was 383±19%
of untreated control. However, the expressions were reduced to 278±79, 259±41,
209±15% of control in DNA treatment groups at concentrations of 2, 4, 8 µM
respectively. But the scramble control did not result in any decrease in the VCAM-1
46
expression, which were 349±24, 360±38 and 393±24% at concentrations of 2, 4, 8 µM
respectively (Fig.15). In TNF-α model, VCAM-1 level was dramatically increased. Since
the signal for media control group was very weak, it is difficult to quantify the
expression. In this case, TNF-α group was used as control group and the results of other
groups were all normalized to that of TNF-α group. GS24-NFκB significantly reduced
the VCAM-1 expression at a concentration of 2 µM, which was 67±8% of TNF-α
control. Scramble ODN had no significant pharmacological effect with VCAM-1 level of
87±11% of control. In addition to protein expression, mRNA expression of VCAM-1 was
also examed. TNF-α dramatically upregulated the mRNA level of VCAM-1, about 19
times of media control. As expected, GS24-NFκB significantly decreased the mRNA
expression to 9±2 times of media control. But the scramble GS24-NFκB did not change
the VCAM-1 expression, which was 18±5 times of media control(Fig.16)
.
47
(a)
(b)
Figure 15 (a) Representative image of western blotting in VCAM-1 expression in OGD/R
model. (b) Effect of GS24-NF-κB on the VCAM-1 expression in OGD/R model, Mean ±
SD, n = 3, ###P < 0.001 vs untreated control, *P< 0.05, ***P< 0.001 vs corresponding
scramble controls.
(a)
48
(b)
med
ia c
ontrol
TNF-
+Scr
amble
contr
ol
TNF-
B
+GS24
-NF
TNF-
0
10
20
30
##
*
VC
AM
-1 m
RN
A e
xp
res
sio
n
(no
rma
lize
d t
o m
ed
ia c
on
tro
l))
(c)
Figure 16 (a) Representative image of western blotting in VCAM-1 expression in TNF-α
model. (b) Effect of GS24-NFκB (2μM) on the VCAM-1 expression in TNF-α model,
Mean ± SD, n = 3-6, **P < 0.01 vs NF-κB decoy treatment and scramble control.(c)
Effect of GS24-NFκB (2μM) on the mRNA expression of VCAM-1 in TNF-α model,
Mean ± SD, n = 3, ##P < 0.01 vs media control, *P <0.05 vs scramble control.
49
5.3.3 GS24-NFκB inhibited ICAM-1 expression in TNF-α model
ICAM-1 is another NF-κB-dependent inflammatory molecule and the effect of GS24-
NFκB on the ICAM-1 expression in TNF-α model was investigated. The results
suggested that GS24-NFκB significantly mitigated the upregualtion of ICAM-1
expression induced by TNF-α. The ICAM-1 level stimulated by TNF-α was 173±23% of
media control but the level was decreased to 122±21% of media control by GS24-NFκB
treatment at a concentration of 2µM. Negative controls including scramble GS24-NFκB
and naked NF-κB decoy did not affect the expression of ICAM-1, in which the ICAM-1
expression were 190±32 and 200±42% of control respectively. RT-PCR results showed
similar mRNA expression profile of ICAM-1. TNF-α enhanced the mRNA level of
ICAM-1, which was 29±5 fold of control. In DNA treatment group, mRNA expression
was only 17±3 fold of control. Scramble ODN and naked NF-κB decoy did not result in
any significant change in ICAM-1 transcript (Fig.17).
(a)
50
(b)
(c)
Figure 17 (a) Representative image of western blotting in ICAM-1 expression in TNF-α
model. (b) Effect of GS24-NFκB (2μM) on the ICAM-1 expression in TNF-α model,
Mean ± SD, n = 3-8, ##P < 0.01 vs media control, **P < 0.01 vs NF-κB or scramble
control. (c) Effect of GS24-NFκB (2μM) on the mRNA expression of ICAM-1 in TNF-
α model, Mean ± SD, n = 3-4, ###P < 0.001 vs media control, **P < 0.01 vs NF-κB decoy
or scramble control.
51
5.3.4 GS24-NFκB inhibited monocyte adhesion to bEND5 cells in TNF-α and
OGD/R inflammatory models
In addition to expression of adhesion molecules, the effect of GS24-NFκB on the
monocyte adhesion was investigated by U937 adhesion assay. When endothelial cells
were stimulated by TNF-α, the percentage of monocyte adhesion to brain endothelium
was increased to 11.3±0.6% compared to non-activated endothelial cells with 6.1±0.6%
adherent monocytes. GS24-NFκB at concentrations of 2 and 4µM reduced the percentage
of adherent U937 cells to 7.7±0.6 and 7.5±0.9% respectively. To exclude any nonspecific
inhibition in adhesion, the experiments were done with scramble GS24-NFκB at
concentrations of 2 and 4 µM, which did not result in significant decrease in adherent
U937 cell with percentage of 11.5±0.7 and 10.5±0.2% respectively (Fig.18a). The
percentage of adherent U937 cells was 5.1±0.2% but the number was significantly
promoted to 9.9±1.1% under OGD/R condition. U937 adhesion was decreased to
5.9±0.6, 6.7±0.5 and 3.9±0.2% with GS24-NFκB treatments at concentrations of 2, 4, 8
µM respectively. Scramble controls at same concentrations did not significantly alter the
U937 adhesion, which were 10.3±0.9, 11.9±0.5 and 9.6±0.8% respectively (Fig.19a). The
representative images were presented in the Fig.18b and Fig.19b.
52
(a)
(b)
Figure 18 (a) Effect of GS24-NFκB on monocytes adhesion to bEND5 cells in TNF-α
model, Mean ± SD, n = 3, ###P < 0.001 vs media control, ***P < 0.001 vs corresponding
scramble controls.(b) Representative images for adhesion assay in TNF-α model (A:
media control; B:TNF-α; C: TNF-α + scramble control(4μM); D: TNF-α + GS24-NFκB
(2μM); E: TNF-α + GS24-NFκB (4μM)).
53
(a)
(b)
Figure 19 (a) Effect of GS24-NFκB on monocytes adhesion to bEND5 cells in OGD/R
model, Mean ± SD, n = 3, ###P < 0.001 vs media control, ***P < 0.001 vs corresponding
scramble controls.(b) Representative images for adhesion assay in OGD/R model (A:
media control; B:OGD/R; C: OGD/R + scramble control(4μM); D: OGD/R + GS24-
NFκB (2μM); E: OGD/R + GS24-NFκB (4μM); F: OGD/R + GS24-NFκB(8μM)).
54
5.4 Pharmacokinetics and organ distribution
The plasma concentration-time profiles for total radioactivity and TCA-precipitable
radioactivity following intravenous injection of GS24-NFκB were shown in Fig.20a. The
plasma concentration-time profiles for TCA-precipitable radioactivity after intravenous
injection of GS24-NFκB and scramble control were shown in Fig.20b. Both plots
indicated a typical two-phase kinetics with an initial rapid distribution phase, followed by
a relatively slow elimination phase. At each time point, the TCA-precipitable
radioactivity was always lower than the total radioactivity and the concentration of GS24-
NFκB was always higher than that of the scramble control. The key pharmacokinetic
parameters were listed in table 1, which indicated very short half-life time 1.92 min and
clearance with 0.35 ml/min.
55
0 20 40 60 800.1
1
10
100 GS24-NFB
TCA-precipitable GS24-NFB
Time(min)
Pla
sm
a c
on
cen
traio
n(
%ID
/ml)
(a)
0 20 40 60 800.1
1
10
100TCA-precipitable ODN
TCA-precipitable scramble ODN
Time(min)
Pla
sm
a c
on
cen
traio
n(
%ID
/ml)
(b)
Figure 20 Plasma concentration time profiles: (a) GS24-NFκB and TCA-precipitable
GS24-NFκB; (b) GS24-NFκB and scramble GS24-NFκB
56
Table 1 Key pharmacokinetic parameters: (a) GS24-NFκB; (b) scramble GS24-NFκB
(a)
Key parameters Unit Mean(SD)
t1/2,α min 1.92(0.16)
t1/2,β min 88.9(43.7)
V ml 1.46(0.10)
Cl ml/min 0.353(0.054)
AUC_60 (%ID/ml)*min 222(15)
(b)
Key parameters Unit Mean(SD)
t1/2,α min 0.79(0.24)
t1/2,β min 21.9(23.8)
V ml 2.07(0.37)
Cl ml/min 1.24(0.15)
AUC_60 (%ID/ml)*min 76(2.3)
The organ distribution for both DNA complex and scramble control groups was indicated
in Fig.21. The brain accumulations for both groups were shown in Fig.21b. In all the
organs, distribution of GS24-NFκB was much higher compared to the scramble control.
The organ accumulations of GS24-NFκB were 7.7, 4.2, 11.1, 9.7, 13.4, 0.39%ID/g for
heart, lung, liver, spleen, kidney and brain respectively.
57
(a)
(b)
Figure 21 Organ distribution of GS24-NFκB and scramble control: (a) heart, lung,
spleen, liver, kidney and brain; (b) brain, Mean ± SD, n = 3, **P < 0.01 vs scramble
control.
5.5 GS24-NFκB inhibited P65 activation and VCAM-1 expression in mouse LPS
model
LPS, a potent inducer of inflammation, was used to produce neuroinflammation within
the brain in the present study. Since the target of the ODN-based delivery system is brain
0
2
4
6
8
10
12
14
16
scra
mb
le
sam
ple
scra
mb
le
sam
ple
scra
mb
le
sam
ple
scra
mb
le
sam
ple
scra
mb
le
sam
ple
scra
mb
le
sam
ple
heart lung liver spleen kidney brain
Org
an d
istr
ibu
tio
n (
%ID
/g)
58
endothelial cells, brain microvessel fraction was isolated and used for protein analysis.
The expressions of phospho-P65 (an important indicator for NF-κB activation) and
VCAM-1 were measured. LPS administration increased the level of phospho-P65 and
VCAM-1 (data did not show here). However, GS24-NFκB had effect on reducing the
phospho-P65 expression (141±26% of media control) compared to that of scramble ODN
(201±34% of media control) (Fig.22). In addition, VCAM-1 expression in scramble
control group was 218±39% of media control while the level was significantly decreased
to 142±29 %of control (Fig.23). The spontaneous locomotor activity significantly
dropped to about 50% of baseline at 16 h following the injection of LPS. However, the
systemic administration of GS24-NFκB and scramble control did not relieve the
suppression of locomotor activity (Fig.24).
59
(a)
med
ia c
ontrol B
LPS+s
cram
ble G
S24
-NF
B
LPS+G
S24
-NF
0
50
100
150
200
250 ###
**
p-p
65 e
xp
ressio
n
(% o
f m
ed
ia c
on
tro
l)
(b)
Figure 22 (a) Representative image of western blotting in phospho-P65 expression in
mouse LPS model.(b) Effect of GS24-NFκB (15mg/kg) on the phospho-P65 level in
mouse LPS model, Mean ± SD, n = 5-6, ###P < 0.001 vs media control, **P < 0.01 vs
scramble control.
60
(a)
med
ia c
ontrol B
LPS+s
cram
ble G
S24
-NF
B
LPS+G
S24
-NF
0
50
100
150
200
250 ###
*
VC
AM
-1 e
xp
ressio
n
(% o
f m
ed
ia c
on
tro
l)
(b)
Figure 23 (a) Representative image of western blotting in VCAM-1 expression in mouse
LPS model.(b) Effect of GS24-NFκB (15mg/kg) on the VCAM-1 level in mouse LPS
model, Mean ± SD, n = 4-5, ###P < 0.001 vs media control, *P < 0.05 vs scramble
control.
61
Figure 24 Effects of DNA complex on sickness behavior induced by systemic
administration of LPS, Mean ± SD, n = 4, **P < 0.01, *P < 0.05 vs baseline
62
CHAPTER 6
DISCUSSION AND CONCLUSION
6.1 The complexity of the role of NF-κB in CNS
Transcription factor NF-κB is a key regulator of a huge array of genes involved in cell
survival and inflammation. It does not only participate in the regulation of physiological
functions in CNS, including neuroprotection in neural development, synaptic
transmission, spatial memory formation and neuroplasticity [190-192], but also act in
stressed or pathological conditions, including cerebral ischemia, multiple sclerosis and
neurodegenerative diseases. Unfortunately, the role of NF-κB in CNS pathologies is not
clear as NF-kB may exert a dual role: protective or detrimental. The “double life” of NF-
κB may challenge our application of NF-κB decoy in CNS diseases to treat
neuroinflammation. Therefore, it is important to discuss the role of NF-κB in CNS first to
justify the therapeutic application in the study.
In cerebral ischemia, many experiments have firmly established that NF-κB is activated
in neurons, endothelial cells, astrocytes and microglia, indicated by its nuclear
translocation, increased DNA binding and transcriptional activity. To elucidate the
function of NF-κB, a range of mouse genetic strategies have been used. The infarct size
was significantly reduced in both transient and permanent stroke models in p50-knockout
mice [193, 194]. Introduction of mutation at the phosphorylation sites of IKK also
suppressed NF-κB activation and thus decreased infarct size and improved neurological
outcome[195].In addition, NF-κB inhibitors such as antioxidants or NBD also showed
63
neuroprotective effects. NF-κB activity was markedly inhibited by pre-treatment with
antioxidants pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC) in the rat
cerebral ischemic model. The two antioxidants also promoted cell survival and reduced
neuronal loss in rat hippocampus [196]. NBD with the function of inhibiting IKK and
block NF-κB activation significantly suppressed the activation of microglia and
ameliorated the disruption of BBB, which suggested the inflammation was inhibited.
Moreover, the treatment improved the neurological scores and reduced cell apoptosis in
rat cerebral ischemia-reperfusion injury [197]. All of the above studies supported that
NF-κB plays a detrimental role in cerebral ischemia. However, controversial reports
indicated that NF-κB contributes to the neuroprotective effect. Duckworth et al. found
that in hippocampus in mouse ischemic brain, neurodegeneration in p50-deficiency mice
was about 8 fold increase relative to non-transgenic mice. And the neurodegeneration
was only confined to the areas with low NF-κB activity[198]. It is well-known that
preconditioning confers resistance to neurons against a subsequent, prolonged ischemia.
And this neuroprotection depended on the activation of NF-κB in neurons in
preconditioning. Inhibition of NF-κB activation at the time of the preconditioning
abolished the protective effect [199].
In conclusion, the role of the NF-κB in CNS pathologies is dependent on context,
including cell types, kinetics of NF-κB activation, insults, combinations of the NF-κB
dimer and interaction with other signaling cascades. The kinetics (timing and duration) of
NF-κB inhibition is critical in determining outcome. As mentioned above, the activation
of NF-κB in preconditioning ischemia was neuroprotective while it was toxic in manifest
ischemia. One explanation is that transient activation induced expression of anti-
64
apoptotic genes but prolonged activation promoted apoptotic and inflammatory genes,
which is consistent with the experiment results obtained by Nijboer et al[200]. They
inhibited NF-κB with NBD coupled to the protein transduction sequence of HIV-TAT
(TAT-NBD) to facilitate cerebral uptake in neonatal hypoxic-ischemic model at different
times and with different duration. Prolonged treatment increased the pro-apoptotic factors
and reduced the anti-apoptotic proteins. In addition, they found out that early NF-κB
activation contributes to the brain damage while late activation protected neurons by up-
regulating anti-apoptotic molecules.
Moreover, NF-κB regulates the patterns of induced genes in a cell-type-specific manner.
NF-κB was found to be constitutively active at high basal levels in neurons and the
constitutive NF-κB activation was required for neuron survival. Inhibition of endogenous
neuronal NF-κB activity in cortical neurons contributed to neuronal death while induction
of NF-κB via overexpression effectively protected primary cortical neurons from death
[201]. Emmanouil et al. generated neuronal IKKβ-deficient mice in MS model, which
showed reduced CNS production of neuroprotective factors and increased production of
proinflammatory cytokines and chemokines. This results supported that neuronal NF-κB
activation is essential to neuroprotection and suppression of inflammation [202]. In
contrast to neurons, no constitutive NF-κB activity is observed in glial cells in normal
condition, which may indicated the different role in these cells. Astrocyte-specific
inhibition of NF-κB protected mice in MS model as expressions of proinflammatory
cytokines and chemokines were suppressed and the expressions of neuroprotective
factors were promoted [203]. On the other hand, a constitutive expression of IKK in
astrocytes induced a widespread neuroinflammation, indicated by the increased
65
expression of inflammatory cytokines and the presence of activated microglia and
astrogliosis [110]. Inhibition of NF-κB by knockout of IKK in microglia significantly
reduced the infarct size in kainic acid challenged mice by decreasing the expression of
inflammatory mediators, such as TNF-α and IL-1β, which indicated activation of NF-κB
contributes to excitotoxin-induced neuronal cell death[204]. Endothelial cell-specific NF-
kB inhibition also relieved inflammation by blocking the expression of inflammatory
molecules [126]. Based on the above previous studies, it is logical to hypothesize that
activation of neuronal NF-κB may promote survival of neurons by inducing the
expression of neuronal anti-apoptotic genes, but activation of endothelial or immune NF-
κB is deleterious in ischemic stroke or other neuroinflammatory diseases by inducing the
synthesis of inflammatory mediators. In the present study, an ODN-based brain drug
delivery system was developed, which consists of NF-κB decoy and GS24 aptamer for
targeted delivery of NF-κB decoy into brain endothelial cells. The GS24-NFκB may
overcome lacking of specificity in NF-κB inhibition and poor efficiency in delivery by
targeting a disease relevant cell population, the brain endothelial cells. Therefore, the
refined approach used in the present study will only inhibit deleterious NF-κB in cerebral
vascular endothelium but leave beneficial NF-κB activity not affected.
6.2 Formation of GS24-NFκB conjugate
The GS24-NFκB is a dual functional DNA complex containing a targeting moiety, GS-
24, for transporting the complex by binding to the TfR, and a DNA decoy for inhibiting
NF-κB activity. As shown in Fig. 4, a completely DNA-based complex has been
synthesized (lane 5). The aptamer GS24 was covalently connected to the decoy ODN via
phosphodiester bond. The GS24 was fused with one strand of the decoy as a whole
66
strand, which was subsequently annealed with the other strand of the decoy to form a
DNA chimera. This resulting linkage promotes the stability of conjugation such that
decoy release during the transport is prevented before it is localized to its cellular target.
The completely DNA-based drug delivery system significantly limits the
immunogenicity. It can be chemically synthesized in large quantities and amenable to
various chemical modifications for improved serum stability and pharmacokinetics. The
size of the DNA complex is relatively small, which promotes tissue penetration compared
to large molecules.
6.3 Cellular uptake of NF-κB
Delivery of GS24-NFκB was investigated in bEnd5 cell as it is a useful in vitro model of
the BBB for drug delivery studies and modeling pathological conditions. A maximal
transendothelial electrical resistance of 121 Omega cm2 was obtained for bEnd5
monolayers. The cells expressed tight junction proteins including ZO-1, occludin and
claudin-1, as well as the transporters P-glycoprotein (P-gp), Na-K-Cl co-transporter
(NKCC), Glucose transporter 1 (GLUT1), and most protein kinase C (PKC) isoforms.
The paracellular and transcellular permeability were investigated by marker permeability
experiments in both normoxia and hypoxia/aglycemia conditions, which indicated that
bEnd5 cells formed a tight barrier and were sensitive to hypoxia/algycemia treatment
[205]. Cell surface receptor, TfR, has been proven to be highly expressed on bEnd5 cells
and the expression was up-regulated in disease conditions [127, 206], which give more
selectivity for our brain gene delivery system. Flow cytometry results confirmed the
delivery capability of GS24-NFκB, which suggested that conjugation with NF-κB decoy
did not affect the conformation and binding ability of GS24. Quantitative uptake of 32P-
67
ODNs demonstrated the delivery specificity as cellular uptake of GS24-NFκB was much
higher relative to scramble control but it was largely reduced in the presence of extra
unlabeled GS24-NFκB in the competition assay. In order to demonstrate that the delivery
was indeed mediated by TfR, TfR was silenced in bEnd5 cells by transfection with anti-
TfR siRNA. We found that the binding and internalization of GS24-NFκB was obviously
decreased in TfR-silenced cells, which indicates that the delivery is not only specific but
also TfR-dependent.
Because of the low permeability of BBB, very little macromolecular cargo is
transcytosed across the cerebral endothelial cells but iron is one of them[207]. TfR has
been proposed to mediate the transcytosis of iron. In addition to brain endothelial cells,
TfRs are also found on the surface of neurons and reactive astrocytes [208-210]. Thus
TfR mediated transcytosis has been explored to deliver therapeutics into brain cells such
as neurons and astrocytes, which has gained some success [211-213]. With respect to the
dual role of NF-κB in CNS, we prefer that our DNA conjugate is only internalized into
brain endothelial cells but not transcytosed into neurons. In the present study, we did not
investigate the transcytosis of the DNA complex as we hypothesize that it is not able to
be delivered across the brain endothelial cells. Many studies have provided evidence that
anti-TfR ligands, especially those with high affinity, were trapped in brain endothelial
cells [214, 215]. Anti-TfR antibodies, OX26, Ri7 and 8D3, intravenously injected into
animals were accumulated in brain endothelial cells and not observed in neurons or
astrocytes. The reasons may be related to the difference of the chemical nature of the
interaction between TfR and either ligands or transferrin and the difference in the
tracking pathways mediated by ligands and transferrin [214, 215]. Bien-Ly et al. found
68
that even anti-TfR antibody affinity determined TfR trafficking fate [213]. After
dissociating from iron, Tf recycled to the plasm while exogenous ligands may not,
especially for aptamers with poor nuclease resistance in cells. It is supposed unable to
transcytosed to the other side of cells and therefore the inhibitory effect of NF-κB is
limited to brain endothelial cells.
6.4 Pharmacological effect in inflammatory models in vitro
The central goal of the present study is to target the endothelial NF-κB by this GS24-
NFκB conjugate for inhibition of vascular inflammation. We have used two in vitro
inflammatory conditions, TNF-α stimulation and OGD/R conditions. TNF-α, an
endogenous cytokine, is able to induce inflammation and NF-κB activation once binding
to its receptor, which has been widely used as in vitro inflammatory model due to the
widespread expression of TNF receptors. OGD/R, modeling hypoxia/reperfusion
ischemic stroke model in vivo, was used as another inflammatory model as the
reperfusion injury significantly induces the activation of NF-κB and promotes the
expression of inflammatory molecules and adhesion molecules[216]. Although NF-κB
has five subunits, the prototypical form is a heterodimer containing binding subunit, P50
and trans-activation subunit, P65[55]. Data from cultured endothelial cells indicate that
P50 and P65 are the predominant species found in nucleus upon cytokine activation [101,
102]. To evaluate the activation of NF-κB, nuclear P65 level was measured as nuclear
localization is a key step in NF-κB activation. In both inflammatory models, GS24-NFκB
chimera significantly reversed the up-regulation of nuclear P65 induced by inflammatory
stimuli, suggesting that the NF-κB activation was suppressed by the treatment of the
DNA complex. To further investigate the transcriptional activity of NF-κB, the protein
69
and mRNA level of NF-κB targeted genes ICAM-1 and VCAM-1 were examed. As
expected, under inflammatory models (TNF-α and OGD/R), mRNA and protein
expressions of both molecules were significantly increased. GS24-NFκB successfully
mitigated the up-regulation and even restored the expressions back to normal level with
high concentrations. This results were consistent with previous studies, which found that
NF-κB decoy have effectively decreased the expression of IL-1α, IL-1β, IL-6, ICAM-1,
and VCAM- 1 at both the transcriptional and translational levels [35, 40, 217]. Since
adhesion molecules are critical in BBB inflammation by mediating leukocytes adhesion
and migration, monocyte (U937) adhesion assay was performed to further verify the anti-
inflammatory effect of the DNA complex. GS24-NFκB inhibited monocyte adhesion to
brain endothelial cells induced by TNFα and OGD/R, which demonstrated that the effect
of the DNA complex at the protein level has been translated into effects at functional
level. The significance of this finding is that GS24-NFκB could be potentially used for
ischemic stroke or other neuroinflammatory diseases therapy.
Although all the above results strongly supported that GS24-NFκB can be effectively
delivered into brain endothelium and inhibit activation and transcriptional activity of NF-
κB, there are several issues needed to be addressed. The first concern is the trafficking
pathway of the DNA conjugate. How did it escaped from the endosomes or lysosomes?
How was it released from the complex? Theoretically, nucleic acids should be degraded
very fast in endosomes or lysosomes with low PH and various nucleases. However, a
number of studies found that aptamer-mediated gene therapy successfully induced
pharmacological effect in specific cells, which implied that they successfully escaped
from endosomes or lysosomes [53, 54, 218]. Unfortunately, the intracellular trafficking
70
path and mechanism of endosomal/lysosomal escape are not clear, which need to be
elucidated in future studies. With respect to the decoy release, there are two possible
explanation. Nucleases randomly cleaved the DNA sequence but some NF-κB decoy
ODNs survived and escaped to induce pharmacological effect. Another possible
mechanism is that the decoy was not released from conjugate and GS24-NFκB as a whole
complex interacted with NF-κB to inhibit its transcriptional activity. The second concern
is about the subcellular localization of the NF-κB-bond decoy: cytoplasm or nucleus.
Generally, molecular weight below 40-60kD is able to pass nuclear pore complex (NPC).
GS24-NFκB with MW of 29.7kD should be allowed to enter nucleus, which is consistent
with our observation in confocal microscope that the DNA complex localized both in
cytoplasm and nucleus (data were not shown here). Previous studies found that delivered
NF-κB decoy predominantly resided in cytoplasm instead of nucleus in airway epithelial
cells while other studies indicated that NF-κB decoy was successfully transfected into
nucleus of hella cells, tracheal epithelial cells (CFTE) and green monkey kidney cells
(Cos 7)[219-221]. The conflictive results suggest that the nucleic uptake of NF-κB is cell
type-specific. Moreover, these studies indicated that the cytoplasmic decoy failed to
inhibit nuclear translocation and transcriptional activity of NF-κB but the nuclear decoy
successfully blocked the NF-κB activation [219-222]. Based on the studies, we
hypothesize that there may be three possible mechanisms underlying the reduced nuclear
level of P65 observed in our study.(1) NF-κB decoy may interact with NF-κB in
cytoplasm and inhibit its nuclear translocation (Fig.3). Although this hypothesis is not
consistent with previous studies indicating that cytoplasmic decoy was not sufficient to
block NF-κB nuclear translocation, the possibility cannot be excluded as even in studies
71
with successful nuclear delivery and NF-κB inhibition, the decoy in nucleus was less than
20% and most of the decoy (more than 80%) was located in cytoplasm[221]. (2) NF-κB
decoys may bind to NF-κB in cytoplasm and then the complex is translocated into
nucleus together. In nucleus, decoy-bound NF-κB cannot interact with target DNA and is
degraded or exported to cytoplasm. (3) NF-κB decoy may enter nucleus and bind to NF-
κB in nucleus, which may induce NF-κB degradation or export to cytoplasm, resulting in
decreased nuclear NF-κB level.
6.5 Pharmacokinetics and biodistribution
To investigate the behavior of the DNA complex in vivo after intravenous administration,
the pharmacokinetic profile and organ distribution were evaluated. The plasma
concentration-time profile showed an expected two-phase kinetics with rapid distribution
phase (t1/2,α, 1.92 min )and slow elimination phase (t1/2,β, 88.9 min). The DNA chimera
was rapidly removed from plasma compartment as about 90% of the injected dose was
removed in the first 5 minutes. The result was consistent with previous studies. The initial
half-life of a biotin-linked oligonucleotide was 1.5 min [184]. In another study NF-κB
decoy was conjugated to PEI/PEG polymers and the half-life of the decoy varied from
1.5 min to 6.5 min[187]. Two key factors may contribute to the rapid removal from
plasma: nuclease-degradation and renal clearance [223]. On the one hand, GS24-NFκB
was rapidly degraded by nucleases based on the % TCA precipitability of plasma [32P]
radioactivity shown in Fig.20a. Total 32P-radioactivity includes both intact and degraded
oligonucleotides while TCA-precipitable radioactivity represents the real plasma
concentration since only TCA-insoluble fraction correlates with undegraded and
minimally degraded ODNs. On the other hand, ODNs was largely cleared by renal
72
infiltration due to their water-soluble properties and the relatively small size (~ 30KD)
[224] and by tubular excretion because of the negative charges on GS24-NFκB inducing
electrostatic clearance across the tubular capillary [225].
As shown in Fig.20 (a), the clearance rate of total samples including degraded and intact
ODNs was lower than that of intact ODNs, which is not consistent with our expectation.
Theoretically, degraded DNAs should show higher clearance rate as their smaller sizes
facilitate the renal clearance. It is hypothesized that the slower clearance rate of the
degraded ODNs results from their binding to plasm protein because binding to plasma
proteins likely prevents glomerular filtration of the oligonucleotides[226]. The hypothesis
is supported by previous studies, which verified the protein binding capability of ODNs
and indicated that the protein binding properties are critical to their distribution and
ultimately the elimination kinetics[226, 227].
Compared to the pharmacokinetic parameters of GS24-NFκB, the scramble control had
reduced the initial half-life (0.79 min), faster clearance and smaller AUC, which may
result from the absence of three-dimensional structure of the scramble control. The
scramble control was designed with the same nucleotide composition but different
sequence. The aptamer has certain folding to facilitate binding to receptor while the
scramble control did not have, which indicated by the electrophoretic gel image of native-
page gel (data were not shown here). The native gel can maintain the secondary structure
and native charge density of the nucleic acids. Although they possess the same molecular
weight, the scramble control migrated faster than GS24-NFκB due to absence of the
folding. GS24-NFκB may have better serum stability as the three-dimensional structure
73
may mask some cleavage-sites of nuclease[228], which may contributes to the longer
half-life and higher AUC value relative to the scramble control.
The highest distribution of GS24-NFκB is in kidney. KANG et al. also reported that
kidney was the organ with highest accumulation with unconjugated ODNs as it was the
principal organ responsible for rapid removal of ODN from plasma. High uptake of
GS24-NFκB was observed in liver and spleen, which was consistent with the organ
distribution of anti-TfR antibody, OX26. The reason contributing to the high uptake may
be the high expression of TfR on the liver and spleen. For all the organs, the uptake of
GS24-NFκB was much higher than that of scramble control, which may results from
specific uptake mediated by TfR and better pharmacokinetic profiles of GS24-NFκB.
Since TfR was widely expressed on almost all the organs, GS24-NFκB could be
specifically taken up by organs via TfR-mediated internalization. The lower clearance
rate and higher AUC of GS24-NFκB relative to the scramble control may be another
reason as organ distribution is directly proportional to the plasma AUC and inversely
related to the rate of plasma clearance[229].
Brain uptake of the DNA chimera was 0.39%ID/g, which was much lower relative to
peripheral organs as expected. To determine whether the brain uptake is high or low,
comparison between the DNA complex and other molecules for brain delivery was made
[230] (Table 2). The brain uptake of GS24-NFκB is comparable to that of another TfR
ligand (0.44%ID/g), OX26, which is anti-TfR monoclonal antibody[229]. OX26
successfully delivered neuroprotective factors into brain and induced neuroprotective
effect [231, 232]. Diazepam is a lipid-soluble small molecule that is nearly 100%
extracted by brain during a single pass through the brain and has no significant efflux
74
[233]. The brain uptake of diazepam in rat is 0.81% ID/g[229], about two folds of that of
GS24-NFκB. The brain uptake of morphine, another small-molecule drug with less lipid
solubility as compared to diazepam, is 0.08% ID/g[234]. However, morphine is a
substrate of P-glycoprotein-mediated efflux system [235, 236]. The brain concentration
was increased by 3 fold in rats treated with P-gp inhibitor [237]. Therefore, the brain
uptake of morphine is about 0.24% without efflux, much less than that of GS24-NFκB.
These comparisons indicate that the brain uptake of GS24-NFκB is acceptable and in the
range that is expected to induce pharmacological effects in CNS following systemic
administration. Not surprisingly, higher brain uptake can be obtained using anti-TfR
antibody. The brain uptake of two anti-TfR monoclonal antibodies 8D3 and RI7-217
were 3.1 and 1.6%ID/g respectively[238]. The higher uptake may result from longer half-
life, lower clearance and enhanced AUC (Table 3) as the brain uptake is directly
proportional to the plasma AUC and inversely related to the rate of plasma
clearance[229]. It is promising that the brain uptake of GS24-NFκB can be further
increased after improving the pharmacokinetics.
Table 2 Brain uptake of GS24-NFκB verus small molecules and TfRMAb
Drug Brain uptake(%ID/g) Animal model
GS24-NFκB 0.39±0.05 mouse
OX26 TfRMAb 0.44±0.07 rat
Diazepan 0.81±0.03 rat
Morphine 0.08±0.007 rat
8D3 TfRMAb 3.1±0.4 mouse
R17 TfRMAb 1.6±0.2 mouse
75
Table 3 Pharmacokinetic parameters for GS24-NFκB and TfRMAbs, 8D3 and R17
Key parameters GS24-NFκB 8D3 R17
t1/2(min) 1.92±0.16 363±26 69±4
Cl(ml/min/kg) 14.12±2.16 0.24±0.03 1.4±0.1
AUC(t)(%ID*min/ml) 222±15 2059±117 1245±133
6.6 Pharmacological effect in vivo
LPS, a major component of the Gram-negative bacteria cell wall, is a potent inducer of
inflammation and now commonly used to produce neuroinflammation as a model for
neurodegenerative diseases [189, 239]. LPS can be recognized by toll-like receptor
(TLR). After binding to TLR, LPS is able to activate NF-κB, resulting in up-regulation
of proinflammatory cytokines[240]. Systemic administration of LPS firstly induced rapid
inflammation in peripheral cavity by triggering release of proinflammatory cytokines,
such as TNF-α, IL-a and inducible isoform of nitric oxide synthase (iNOS) [241, 242].
These cytokines can be transported across BBB and activate microglia and endothelial
cells, leading to inflammatory responses in brain and reduced locomotor activity [243-
246].
In current study, systemic administration of LPS (1mg/kg, i.p) was performed to
generate neuroinflammatory model to evaluate the therapeutic effect of GS24-NFκB. As
expected, LPS suppressed the spontaneous locomotor activity to about 50% of the
baseline. Then the activation of NF-κB and the expression of its targeted gene VCAM-1
were investigated. Although nuclear translocation of NF-κB is critical for its activation,
recent evidence suggested that it was not sufficient to activate NF-κB-dependent
transcription. Phosphorylation of P65 is involved in eliciting a maximal NF-κB response
[109, 247]. Yang et al. found that LPS induced phosphorylation of the P65 on serine 536
76
and the phospho-P65 (p-P65) effectively enhanced transcriptional activity [247].
Consistent with the previous findings, systemic administration of LPS effectively
increased the level of p-P65 (Ser536) and VCAM-1 in brain microvessel. It is well-
known that the therapeutic use of ODNs is strongly limited by two problems. One is cell
delivery since the macromolecular, polyanionic and highly hydrophilic nature of ODNs
hampers their cellular uptake. In the present study, GS24 aptamer successfully solved this
problem by taking advantage of receptor-mediated transport system on cell surface to
facilitate cell-specific delivery. The other problem is the pharmacokinetics of ODNs with
poor serum stability and rapid clearance. However, to our surprise, the inhibitory effects
of systemic administration of GS24-NFκB on the expression of phospho-P65 and
VCAM-1 were observed in LPS-challenged mice while the therapeutic effect in behavior
test was not observed. We hypothesized that most of the administered GS24-NFκB was
degraded or cleared in circulation and only a little amount finally reached its target, brain
endothelium. Therefore, with high dose injected, the pharmacological effect on the
protein expression can be detected in microvessel but the amount is not sufficient to
induce disease-modifying effect. These results suggest that this novel ODN delivery
system is promising in the clinical application of treating neuroinflammation once
pharmacokinetics is improved.
6.7 Conclusion
In conclusion, the present study has synthesized an aptamer-therapeutic ODN conjugate
(GS24-NFκB) for brain delivery of NF-κB decoy with the goal to inhibit
neuroinflammation. The DNA complex was specifically taken up by bEnd5 cells via
TfR-mediated endocytosis. In TNF-α and OGD/R induced inflammatory model in vitro,
77
GS24-NFκB significantly reduced nuclear level of P65, indicating inhibition of NF-κB
activation. The transcriptional activity of NF-κB was effectively suppressed by GS24-
NFκB, indicated by decreased expression of NF-κB-dependent inflammatory genes,
ICAM-1 and VCAM-1. Moreover, the DNA complex largely inhibited the monocyte
adhesion to brain endothelial cells induced by TNF-α or OGD/R, which verified anti-
inflammatory effect of GS24-NFκB. Pharmacokinetic study suggested that the DNA
complex was susceptible to nucleases and removed rapidly from plasma with short half-
life. The DNA complex can be specifically taken up by TfR-expressing cells as high
distribution was observed in TfR highly expressed organs, spleen and liver. The
accumulation of GS24-NFκB in brain was about 0.4%ID/g, which was significantly
higher than that of scramble control. The brain uptake value is high enough to elicit
pharmacological effects in CNS, which was supported by our in vivo study. GS24-NFκB
significantly reversed the up-regulation of p-P65 and VCAM-1 in mouse microvessels
induced by LPS. This study provides a proof of concept for brain-targeted drug delivery
and a refined approach to target and modulate a particular cell population, the brain
endothelial cells, relevant to the disease. The study explores the cerebral endothelial cell
as a novel site of action for brain drug delivery and neuro-vascular inflammation therapy.
78
CHAPTER 7
FUTURE DIRECTIONS
As a proof of concept study, we have demonstrated the potential of GS24-NFκB in brain
delivery and inhibition of neuro-vascular inflammation. However, there is a long way for
clinical application. Future work will focus on the issues mentioned in the discussion
section, including subcellular localization of the decoy, intracellular trafficking pathway
and improvement of pharmacokinetics and therapeutic effects in vivo.
7.1 Intracellular trafficking path and subcellular localization of GS24-NFκB
Although GS24-NFκB indeed induced pharmacological effect in cells, two questions are
needed to be answered for verification of its effect. First, although transcription factor
decoys have been widely used in intervention of many diseases and its potential
therapeutic application has been proven [35, 40, 127], the mechanisms underlying how it
works in cells have not been well studied. Whether the inhibitory effect on NF-κB is
resulted from interaction of the decoy with cytoplasmic or nuclear NF-κB is not clear.
Therefore, in our future studies, the subcellular localization of decoy-bound NF-κB will
be investigated for better understanding the behavior of the decoy in cells. Secondly,
lysosomes or endosomes have been demonstrated to be involved in the TfR-mediated
endocytosis, in which the nucleases easily degraded the DNA complex. However, many
studies including the present study suggested the decoy should successfully escape from
endosomes or lysosomes as pharmacological effects were observed [248]. Therefore, the
79
intracellular trafficking path of the DNA complex will be investigated to figure out the
mechanism of endosomal/lysosomal escape.
The bEnd5 cells will be incubated with fluorescent labeled GS24-NFκB. After incubation
at 37°C for different time periods, cells will be fixed by 4% paraformaldehyde and
stained with appropriate antibodies which are used as markers for subcellular
compartment: anti-EEA1 for early endosomes; anti-Rab4 for recycling endosomes; anti-
Rab7 for late endosomes; anti-lamp-1 for lysosomes. Moreover, Topro-3 iodine and
Alexa Fluor 488 phalloidin will counter-stain nuclei and actin. The subcellular
compartments involved in the uptake and trafficking will be identified by colocalization
of DNA or RNA chimeras with these markers. Scramble GS24-NFκB with scramble
GS24 will serve as negative controls.
7.2 Chemical modification of GS24-NFκB
The pharmacokinetic profile of GS24-NFκB indicated poor stability in blood and rapid
clearance from circulation, which represent the largest historical obstacle to widespread
use of aptamer-mediated gene therapy. One strategy that has been widely used to increase
the serum stability of ODNs is phosphorothioate (PS) modification, which involves
replacement of non-bridging oxygen atoms in the phosphate group with sulfur [249, 250].
The phosphorothioate modification not only enhanced the resistance to nucleases
compared with phosphodiester (DNA) but also improved distribution and
pharmacokinetics overall[251]. PS ODNs have higher binding affinity to plasma proteins,
which may reduce the renal clearance and improve the pharmacokinetics and
distribution[226]. However, attention should be paid to the toxicity of PS ODNs[252].
Partially thiophosphorylated instead of complete substitution is preferred to maximally
80
enhance the stability and limit the toxicity[251]. Blockage of the 3' or 5' ends of aptamers
with small molecules is another strategy, which successfully reduced enzymatic
degradation. 3’end capping strategy with inverted thymidine inhibits the predominant
nuclease degradation in serum, 3’ exonuclease attack and increases the serum half-life
[253, 254]. In addition, 3’-biotin-streptavidin conjugated ODNs have higher resistance to
3’ exonuclease and lower clearance in systemic circulation [253, 255]. Conjugation of
ODNs with polyethylene glycol (PEG) significantly reduces the systemic clearance by
increasing the molecular size [256, 257].
The pharmacokinetics of the modified ODNs will be investigated to verify whether the
modifications improve the biostability and enhance the brain uptake of the DNA
complex. Attention should be paid to the efficacy of the modified DNA chimera as the
binding affinity and specificity of the aptamer may be affected by the modified sugars
especially if internal to the aptamer chain and associated with a binding site[164].
7.3 Therapeutic effect in vivo
The therapeutic effect in inhibition of neuroinflammation will be further evaluated. The
number of the animals involved in behavior test is only four, which is not enough to
obtain a reliable result. More animals will be tested in this experiments. To verify the
specific inhibitory effect on NF-κB, another scramble control with GS24 and scramble
decoy sequence will be used in future study. To evaluate the neuroprotective effect of the
DNA complex, a variety of experiments can be applied, including assessment of
neurological deficit, infiltration of immune cells and BBB permeability. To investigate
the toxicity of the DNA complex, one control group with only DNA injection without
LPS challenge will be added.
81
Although TfR has been demonstrated to allow noninvasive delivery of various
therapeutic agents to the brain, the ubiquitous expression of the TfR on peripheral organs
limits its capability for specific brain delivery and induces side effects in non-brain
tissues. More specific brain delivery strategy should be developed. Comparison between
two TfRMAbs, 8D3 and R17, indicated that R17 had better brain specificity as there was
no uptake in mouse liver[238]. R17 is hypothesized to bind to an epitope that is fully
expressed at the BBB TfR, but is masked in part at the TfR in mouse liver. It is very
important to identify such brain-specific epitope and develop aptamers targeting them for
brain delivery, which will largely improve the therapeutic effect of aptamer-mediated
gene therapy.
82
REFERENCES
1. Neurological Conditions 2014; Available from:
http://www.movementforhope.org/what-is-a-neurological-disease/.
2. de Araujo, E.G., G.M. da Silva, and A.A. Dos Santos, Neuronal cell survival: the
role of interleukins. Ann N Y Acad Sci, 2009. 1153: p. 57-64.
3. Kerschensteiner, M., et al., Activated human T cells, B cells, and monocytes
produce brain-derived neurotrophic factor in vitro and in inflammatory brain
lesions: a neuroprotective role of inflammation? J Exp Med, 1999. 189(5): p.
865-70.
4. Harry, G.J. and A.D. Kraft, Neuroinflammation and microglia: considerations
and approaches for neurotoxicity assessment. Expert Opin Drug Metab Toxicol,
2008. 4(10): p. 1265-77.
5. Lucas, S.M., N.J. Rothwell, and R.M. Gibson, The role of inflammation in CNS
injury and disease. Br J Pharmacol, 2006. 147 Suppl 1: p. S232-40.
6. Minghetti, L., Role of inflammation in neurodegenerative diseases. Curr Opin
Neurol, 2005. 18(3): p. 315-21.
7. Hirsch, E.C. and S. Hunot, Neuroinflammation in Parkinson's disease: a target
for neuroprotection? Lancet Neurol, 2009. 8(4): p. 382-97.
8. Nataf, S., Neuroinflammation responses and neurodegeneration in multiple
sclerosis. Rev Neurol (Paris), 2009. 165(12): p. 1023-8.
9. Naegele, M. and R. Martin, The good and the bad of neuroinflammation in
multiple sclerosis. Handb Clin Neurol, 2014. 122: p. 59-87.
10. Shah, I.M., I.M. Macrae, and M. Di Napoli, Neuroinflammation and
neuroprotective strategies in acute ischaemic stroke - from bench to bedside. Curr
Mol Med, 2009. 9(3): p. 336-54.
11. de Vries, H.E., et al., The blood-brain barrier in neuroinflammatory diseases.
Pharmacol Rev, 1997. 49(2): p. 143-55.
12. Webb, A.A. and G.D. Muir, The blood-brain barrier and its role in inflammation.
J Vet Intern Med, 2000. 14(4): p. 399-411.
13. Lacroix, S., D. Feinstein, and S. Rivest, The bacterial endotoxin
lipopolysaccharide has the ability to target the brain in upregulating its
membrane CD14 receptor within specific cellular populations. Brain Pathol,
1998. 8(4): p. 625-40.
14. Quan, N., M. Whiteside, and M. Herkenham, Time course and localization
patterns of interleukin-1beta messenger RNA expression in brain and pituitary
after peripheral administration of lipopolysaccharide. Neuroscience, 1998. 83(1):
p. 281-93.
15. Barone, F.C., et al., Tumor necrosis factor-alpha. A mediator of focal ischemic
brain injury. Stroke, 1997. 28(6): p. 1233-44.
16. Nadeau, S. and S. Rivest, Regulation of the gene encoding tumor necrosis factor
alpha (TNF-alpha) in the rat brain and pituitary in response in different models
of systemic immune challenge. J Neuropathol Exp Neurol, 1999. 58(1): p. 61-77.
83
17. Yang, G.Y., et al., Tumor necrosis factor alpha expression produces increased
blood-brain barrier permeability following temporary focal cerebral ischemia in
mice. Brain Res Mol Brain Res, 1999. 69(1): p. 135-43.
18. Quan, N., M. Whiteside, and M. Herkenham, Cyclooxygenase 2 mRNA expression
in rat brain after peripheral injection of lipopolysaccharide. Brain Res, 1998.
802(1-2): p. 189-97.
19. Wong, M.L., et al., Inducible nitric oxide synthase gene expression in the brain
during systemic inflammation. Nat Med, 1996. 2(5): p. 581-4.
20. Lindsberg, P.J., et al., Endothelial ICAM-1 expression associated with
inflammatory cell response in human ischemic stroke. Circulation, 1996. 94(5): p.
939-45.
21. Henninger, D.D., et al., Cytokine-induced VCAM-1 and ICAM-1 expression in
different organs of the mouse. J Immunol, 1997. 158(4): p. 1825-32.
22. Stanimirovic, D.B., et al., Increase in surface expression of ICAM-1, VCAM-1
and E-selectin in human cerebromicrovascular endothelial cells subjected to
ischemia-like insults. Acta Neurochir Suppl, 1997. 70: p. 12-6.
23. de Vries, H.E., et al., Effect of endotoxin on permeability of bovine cerebral
endothelial cell layers in vitro. J Pharmacol Exp Ther, 1996. 277(3): p. 1418-23.
24. de Vries, H.E., et al., The influence of cytokines on the integrity of the blood-brain
barrier in vitro. J Neuroimmunol, 1996. 64(1): p. 37-43.
25. Aloisi, F., Immune function of microglia. Glia, 2001. 36(2): p. 165-79.
26. Nakajima, K. and S. Kohsaka, Microglia: activation and their significance in the
central nervous system. J Biochem, 2001. 130(2): p. 169-75.
27. Baldwin, A.S., Jr., Series introduction: the transcription factor NF-kappaB and
human disease. J Clin Invest, 2001. 107(1): p. 3-6.
28. Keifer, J.A., et al., Inhibition of NF-kappa B activity by thalidomide through
suppression of IkappaB kinase activity. J Biol Chem, 2001. 276(25): p. 22382-7.
29. Lawrence, T., The nuclear factor NF-kappaB pathway in inflammation. Cold
Spring Harb Perspect Biol, 2009. 1(6): p. a001651.
30. Kooij, G., et al., T lymphocytes impair P-glycoprotein function during
neuroinflammation. J Autoimmun. 34(4): p. 416-25.
31. Pan, W., et al., The role of cerebral vascular NFkappaB in LPS-induced
inflammation: differential regulation of efflux transporter and transporting
cytokine receptors. Cell Physiol Biochem. 25(6): p. 623-30.
32. Quan, N., et al., Induction of inhibitory factor kappaBalpha mRNA in the central
nervous system after peripheral lipopolysaccharide administration: an in situ
hybridization histochemistry study in the rat. Proc Natl Acad Sci U S A, 1997.
94(20): p. 10985-90.
33. Laflamme, N., S. Lacroix, and S. Rivest, An essential role of interleukin-1beta in
mediating NF-kappaB activity and COX-2 transcription in cells of the blood-
brain barrier in response to a systemic and localized inflammation but not during
endotoxemia. J Neurosci, 1999. 19(24): p. 10923-30.
34. Bielinska, A., et al., Regulation of gene expression with double-stranded
phosphorothioate oligonucleotides. Science, 1990. 250(4983): p. 997-1000.
84
35. Morishita, R., et al., In vivo transfection of cis element "decoy" against nuclear
factor-kappaB binding site prevents myocardial infarction. Nat Med, 1997. 3(8):
p. 894-9.
36. Tomita, N., et al., Transcription factor decoy for NFkappaB inhibits TNF-alpha-
induced cytokine and adhesion molecule expression in vivo. Gene Ther, 2000.
7(15): p. 1326-32.
37. Tomita, N., et al., Inhibition of TNF-alpha, induced cytokine and adhesion
molecule. Expression in glomerular cells in vitro and in vivo by transcription
factor decoy for NFkappaB. Exp Nephrol, 2001. 9(3): p. 181-90.
38. Matsuda, N., et al., Therapeutic effect of in vivo transfection of transcription
factor decoy to NF-kappaB on septic lung in mice. Am J Physiol Lung Cell Mol
Physiol, 2004. 287(6): p. L1248-55.
39. Xu, J., et al., Regulation of cytokine-induced iNOS expression by a hairpin
oligonucleotide in murine cerebral endothelial cells. Biochem Biophys Res
Commun, 1997. 235(2): p. 394-7.
40. Tomita, N., et al., Transcription factor decoy for nuclear factor-kappaB inhibits
tumor necrosis factor-alpha-induced expression of interleukin-6 and intracellular
adhesion molecule-1 in endothelial cells. J Hypertens, 1998. 16(7): p. 993-1000.
41. Hess, D.C., et al., Hypertonic mannitol loading of NF-kappaB transcription factor
decoys in human brain microvascular endothelial cells blocks upregulation of
ICAM-1. Stroke, 2000. 31(5): p. 1179-86.
42. Bickel, U., T. Yoshikawa, and W.M. Pardridge, Delivery of peptides and proteins
through the blood-brain barrier. Adv Drug Deliv Rev, 2001. 46(1-3): p. 247-79.
43. Chen, C.H., et al., Aptamer-based endocytosis of a lysosomal enzyme. Proc Natl
Acad Sci U S A, 2008. 105(41): p. 15908-13.
44. Brusaferri, F. and L. Candelise, Steroids for multiple sclerosis and optic neuritis:
a meta-analysis of randomized controlled clinical trials. J Neurol, 2000. 247(6):
p. 435-42.
45. Bauer, B., et al., Pregnane X receptor up-regulation of P-glycoprotein expression
and transport function at the blood-brain barrier. Mol Pharmacol, 2004. 66(3): p.
413-9.
46. Scott, S., et al., Design, power, and interpretation of studies in the standard
murine model of ALS. Amyotroph Lateral Scler, 2008. 9(1): p. 4-15.
47. Cudkowicz, M.E., et al., Trial of celecoxib in amyotrophic lateral sclerosis. Ann
Neurol, 2006. 60(1): p. 22-31.
48. Jaturapatporn, D., et al., Aspirin, steroidal and non-steroidal anti-inflammatory
drugs for the treatment of Alzheimer's disease. Cochrane Database Syst Rev,
2012. 2: p. CD006378.
49. Hoppmann, R.A., J.G. Peden, and S.K. Ober, Central nervous system side effects
of nonsteroidal anti-inflammatory drugs. Aseptic meningitis, psychosis, and
cognitive dysfunction. Arch Intern Med, 1991. 151(7): p. 1309-13.
50. Auriel, E., K. Regev, and A.D. Korczyn, Nonsteroidal anti-inflammatory drugs
exposure and the central nervous system. Handb Clin Neurol, 2014. 119: p. 577-
84.
51. Zhou, J. and J.J. Rossi, Aptamer-targeted cell-specific RNA interference. Silence,
2010. 1(1): p. 4.
85
52. Zhou, J., et al., Current progress of RNA aptamer-based therapeutics. Front
Genet, 2012. 3: p. 234.
53. McNamara, J.O., 2nd, et al., Cell type-specific delivery of siRNAs with aptamer-
siRNA chimeras. Nat Biotechnol, 2006. 24(8): p. 1005-15.
54. Shu, D., et al., Construction of phi29 DNA-packaging RNA monomers, dimers,
and trimers with variable sizes and shapes as potential parts for nanodevices. J
Nanosci Nanotechnol, 2003. 3(4): p. 295-302.
55. Oeckinghaus, A. and S. Ghosh, The NF-kappaB family of transcription factors
and its regulation. Cold Spring Harb Perspect Biol, 2009. 1(4): p. a000034.
56. Piskunov, A.K., Neuroinflammation biomarkers. Neurochemical Journal, 2010.
4(1): p. 55-63.
57. Infante-Duarte, C., et al., New developments in understanding and treating
neuroinflammation. J Mol Med (Berl), 2008. 86(9): p. 975-85.
58. Bechmann, I., I. Galea, and V.H. Perry, What is the blood-brain barrier (not)?
Trends Immunol, 2007. 28(1): p. 5-11.
59. Weinstein, J.R., I.P. Koerner, and T. Moller, Microglia in ischemic brain injury.
Future Neurol, 2010. 5(2): p. 227-246.
60. McGeer, P.L., et al., Reactive microglia in patients with senile dementia of the
Alzheimer type are positive for the histocompatibility glycoprotein HLA-DR.
Neurosci Lett, 1987. 79(1-2): p. 195-200.
61. Rogers, J., et al., Expression of immune system-associated antigens by cells of the
human central nervous system: relationship to the pathology of Alzheimer's
disease. Neurobiol Aging, 1988. 9(4): p. 339-49.
62. Imamura, K., et al., Distribution of major histocompatibility complex class II-
positive microglia and cytokine profile of Parkinson's disease brains. Acta
Neuropathol, 2003. 106(6): p. 518-26.
63. Harling-Berg, C., et al., Role of cervical lymph nodes in the systemic humoral
immune response to human serum albumin microinfused into rat cerebrospinal
fluid. J Neuroimmunol, 1989. 25(2-3): p. 185-93.
64. Joo, F., The cerebral microvessels in culture, an update. J Neurochem, 1992.
58(1): p. 1-17.
65. Bradbury, M.W., The blood-brain barrier. Transport across the cerebral
endothelium. Circ Res, 1985. 57(2): p. 213-22.
66. Anderson, J.M. and C.M. Van Itallie, Physiology and function of the tight
junction. Cold Spring Harb Perspect Biol, 2009. 1(2): p. a002584.
67. Butt, A.M., H.C. Jones, and N.J. Abbott, Electrical resistance across the blood-
brain barrier in anaesthetized rats: a developmental study. J Physiol, 1990. 429:
p. 47-62.
68. Hawkins, B.T. and T.P. Davis, The blood-brain barrier/neurovascular unit in
health and disease. Pharmacol Rev, 2005. 57(2): p. 173-85.
69. Cervos-Navarro, J., S. Kannuki, and Y. Nakagawa, Blood-brain barrier (BBB).
Review from morphological aspect. Histol Histopathol, 1988. 3(2): p. 203-13.
70. Cordon-Cardo, C., et al., Multidrug-resistance gene (P-glycoprotein) is expressed
by endothelial cells at blood-brain barrier sites. Proc Natl Acad Sci U S A, 1989.
86(2): p. 695-8.
86
71. Abbott, N.J., L. Ronnback, and E. Hansson, Astrocyte-endothelial interactions at
the blood-brain barrier. Nat Rev Neurosci, 2006. 7(1): p. 41-53.
72. Cornford, E.M. and S. Hyman, Blood-brain barrier permeability to small and
large molecules. Adv Drug Deliv Rev, 1999. 36(2-3): p. 145-163.
73. Scheld, W.M., Drug delivery to the central nervous system: general principles
and relevance to therapy for infections of the central nervous system. Rev Infect
Dis, 1989. 11 Suppl 7: p. S1669-90.
74. Barker, C.F. and R.E. Billingham, Immunologically privileged sites. Adv
Immunol, 1977. 25: p. 1-54.
75. Pardridge, W.M., Blood-brain barrier transport of glucose, free fatty acids, and
ketone bodies. Adv Exp Med Biol, 1991. 291: p. 43-53.
76. Visser, C.C., et al., Characterization and modulation of the transferrin receptor
on brain capillary endothelial cells. Pharm Res, 2004. 21(5): p. 761-9.
77. Schinkel, A.H., P-Glycoprotein, a gatekeeper in the blood-brain barrier. Adv
Drug Deliv Rev, 1999. 36(2-3): p. 179-194.
78. Nagyoszi, P., et al., Expression and regulation of toll-like receptors in cerebral
endothelial cells. Neurochem Int, 2010. 57(5): p. 556-64.
79. Etienne, S., et al., MHC class II engagement in brain endothelial cells induces
protein kinase A-dependent IL-6 secretion and phosphorylation of cAMP
response element-binding protein. J Immunol, 1999. 163(7): p. 3636-41.
80. Pan, W., et al., Cytokine signaling modulates blood-brain barrier function. Curr
Pharm Des, 2011. 17(33): p. 3729-40.
81. Osburg, B., et al., Effect of endotoxin on expression of TNF receptors and
transport of TNF-alpha at the blood-brain barrier of the rat. Am J Physiol
Endocrinol Metab, 2002. 283(5): p. E899-908.
82. Van Dam, A.M., et al., Interleukin-1 receptors on rat brain endothelial cells: a
role in neuroimmune interaction? FASEB J, 1996. 10(2): p. 351-6.
83. Erickson, M.A., K. Dohi, and W.A. Banks, Neuroinflammation: a common
pathway in CNS diseases as mediated at the blood-brain barrier.
Neuroimmunomodulation, 2012. 19(2): p. 121-30.
84. Deli, M.A., et al., Exposure of tumor necrosis factor-alpha to luminal membrane
of bovine brain capillary endothelial cells cocultured with astrocytes induces a
delayed increase of permeability and cytoplasmic stress fiber formation of actin. J
Neurosci Res, 1995. 41(6): p. 717-26.
85. Duchini, A., et al., Effects of tumor necrosis factor-alpha and interleukin-6 on
fluid-phase permeability and ammonia diffusion in CNS-derived endothelial cells.
J Investig Med, 1996. 44(8): p. 474-82.
86. Springer, T.A., Adhesion receptors of the immune system. Nature, 1990.
346(6283): p. 425-34.
87. Bevilacqua, M.P. and R.M. Nelson, Selectins. J Clin Invest, 1993. 91(2): p. 379-
87.
88. Hynes, R.O., Integrins: versatility, modulation, and signaling in cell adhesion.
Cell, 1992. 69(1): p. 11-25.
89. Osborn, L., Leukocyte adhesion to endothelium in inflammation. Cell, 1990.
62(1): p. 3-6.
87
90. Kebir, H., et al., Human TH17 lymphocytes promote blood-brain barrier
disruption and central nervous system inflammation. Nat Med, 2007. 13(10): p.
1173-5.
91. Engelhardt, B. and H. Wolburg, Mini-review: Transendothelial migration of
leukocytes: through the front door or around the side of the house? Eur J
Immunol, 2004. 34(11): p. 2955-63.
92. del Zoppo, G.J., The neurovascular unit, matrix proteases, and innate
inflammation. Ann N Y Acad Sci, 2010. 1207: p. 46-9.
93. Rosenberg, G.A., Matrix metalloproteinases in brain injury. J Neurotrauma,
1995. 12(5): p. 833-42.
94. Chandler, S., et al., Matrix metalloproteinases, tumor necrosis factor and multiple
sclerosis: an overview. J Neuroimmunol, 1997. 72(2): p. 155-61.
95. Nagase, H. and J.F. Woessner, Jr., Matrix metalloproteinases. J Biol Chem, 1999.
274(31): p. 21491-4.
96. Kontos, H.A., et al., Appearance of superoxide anion radical in cerebral
extracellular space during increased prostaglandin synthesis in cats. Circ Res,
1985. 57(1): p. 142-51.
97. Feuerstein, G. and J.M. Hallenbeck, Leukotrienes in health and disease. FASEB
J, 1987. 1(3): p. 186-92.
98. Morin, A.M. and A. Stanboli, Nitric oxide synthase localization in cultured
cerebrovascular endothelium during mitosis. Exp Cell Res, 1994. 211(2): p. 183-
8.
99. Ridder, D.A. and M. Schwaninger, NF-kappaB signaling in cerebral ischemia.
Neuroscience, 2009. 158(3): p. 995-1006.
100. Haddad, J.J. and N.E. Abdel-Karim, NF-kappaB cellular and molecular
regulatory mechanisms and pathways: therapeutic pattern or pseudoregulation?
Cell Immunol, 2011. 271(1): p. 5-14.
101. Collins, T., et al., Transcriptional regulation of endothelial cell adhesion
molecules: NF-kappa B and cytokine-inducible enhancers. FASEB J, 1995. 9(10):
p. 899-909.
102. Pober, J.S., Endothelial activation: intracellular signaling pathways. Arthritis
Res, 2002. 4 Suppl 3: p. S109-16.
103. Malek, S., et al., X-ray crystal structure of an IkappaBbeta x NF-kappaB p65
homodimer complex. J Biol Chem, 2003. 278(25): p. 23094-100.
104. Huxford, T., et al., The crystal structure of the IkappaBalpha/NF-kappaB complex
reveals mechanisms of NF-kappaB inactivation. Cell, 1998. 95(6): p. 759-70.
105. Mc Guire, C., et al., Nuclear factor kappa B (NF-kappaB) in multiple sclerosis
pathology. Trends Mol Med, 2013. 19(10): p. 604-13.
106. Zandi, E., Y. Chen, and M. Karin, Direct phosphorylation of IkappaB by
IKKalpha and IKKbeta: discrimination between free and NF-kappaB-bound
substrate. Science, 1998. 281(5381): p. 1360-3.
107. Chen, Z.J., L. Parent, and T. Maniatis, Site-specific phosphorylation of
IkappaBalpha by a novel ubiquitination-dependent protein kinase activity. Cell,
1996. 84(6): p. 853-62.
108. Chen, L.F., et al., NF-kappaB RelA phosphorylation regulates RelA acetylation.
Mol Cell Biol, 2005. 25(18): p. 7966-75.
88
109. Perkins, N.D., Post-translational modifications regulating the activity and
function of the nuclear factor kappa B pathway. Oncogene, 2006. 25(51): p. 6717-
30.
110. Oeckl, P., et al., Astrocyte-specific IKK2 activation in mice is sufficient to induce
neuroinflammation but does not increase susceptibility to MPTP. Neurobiol Dis,
2012. 48(3): p. 481-7.
111. Hayden, M.S. and S. Ghosh, Signaling to NF-kappaB. Genes Dev, 2004. 18(18):
p. 2195-224.
112. Kiernan, R., et al., Post-activation turn-off of NF-kappa B-dependent
transcription is regulated by acetylation of p65. J Biol Chem, 2003. 278(4): p.
2758-66.
113. Flood, P.M., et al., Transcriptional Factor NF-kappaB as a Target for Therapy in
Parkinson's Disease. Parkinsons Dis, 2011. 2011: p. 216298.
114. Yan, J. and J.M. Greer, NF-kappa B, a potential therapeutic target for the
treatment of multiple sclerosis. CNS Neurol Disord Drug Targets, 2008. 7(6): p.
536-57.
115. Wen, Y., et al., Estrogen attenuates nuclear factor-kappa B activation induced by
transient cerebral ischemia. Brain Res, 2004. 1008(2): p. 147-54.
116. Pereira, M.P., et al., Rosiglitazone and 15-deoxy-Delta12,14-prostaglandin J2
cause potent neuroprotection after experimental stroke through noncompletely
overlapping mechanisms. J Cereb Blood Flow Metab, 2006. 26(2): p. 218-29.
117. Nagashima, K., et al., Rapid TNFR1-dependent lymphocyte depletion in vivo with
a selective chemical inhibitor of IKKbeta. Blood, 2006. 107(11): p. 4266-73.
118. Gveric, D., et al., Transcription factor NF-kappaB and inhibitor I kappaBalpha
are localized in macrophages in active multiple sclerosis lesions. J Neuropathol
Exp Neurol, 1998. 57(2): p. 168-78.
119. Bonetti, B., et al., Activation of NF-kappaB and c-jun transcription factors in
multiple sclerosis lesions. Implications for oligodendrocyte pathology. Am J
Pathol, 1999. 155(5): p. 1433-8.
120. Mycko, M.P., et al., cDNA microarray analysis in multiple sclerosis lesions:
detection of genes associated with disease activity. Brain, 2003. 126(Pt 5): p.
1048-57.
121. Whitney, L.W., et al., Analysis of gene expression in mutiple sclerosis lesions
using cDNA microarrays. Ann Neurol, 1999. 46(3): p. 425-8.
122. Heck, S., et al., I kappaB alpha-independent downregulation of NF-kappaB
activity by glucocorticoid receptor. EMBO J, 1997. 16(15): p. 4698-707.
123. Scheinman, R.I., et al., Role of transcriptional activation of I kappa B alpha in
mediation of immunosuppression by glucocorticoids. Science, 1995. 270(5234): p.
283-6.
124. Schweingruber, N., et al., Mechanisms of glucocorticoids in the control of
neuroinflammation. J Neuroendocrinol, 2012. 24(1): p. 174-82.
125. Ghosh, A., et al., Selective inhibition of NF-kappaB activation prevents
dopaminergic neuronal loss in a mouse model of Parkinson's disease. Proc Natl
Acad Sci U S A, 2007. 104(47): p. 18754-9.
89
126. Fischer, D., et al., Inhibition of monocyte adhesion on brain-derived endothelial
cells by NF-kappaB decoy/polyethylenimine complexes. J Gene Med, 2005. 7(8):
p. 1063-76.
127. Bhattacharya, R., et al., Targeted delivery of complexes of biotin-PEG-
polyethylenimine and NF-kappaB decoys to brain-derived endothelial cells in
vitro. Pharm Res, 2008. 25(3): p. 605-15.
128. Goodchild, J., Therapeutic oligonucleotides. Methods Mol Biol, 2011. 764: p. 1-
15.
129. Grillone, L.R. and R. Lanz, Fomivirsen. Drugs Today (Barc), 2001. 37(4): p. 245-
255.
130. Mann, M.J. and V.J. Dzau, Therapeutic applications of transcription factor decoy
oligonucleotides. J Clin Invest, 2000. 106(9): p. 1071-5.
131. Mann, M.J., Transcription factor decoys: a new model for disease intervention.
Ann N Y Acad Sci, 2005. 1058: p. 128-39.
132. Morishita, R., et al., A gene therapy strategy using a transcription factor decoy of
the E2F binding site inhibits smooth muscle proliferation in vivo. Proc Natl Acad
Sci U S A, 1995. 92(13): p. 5855-9.
133. Park, Y.G., et al., Dual blockade of cyclic AMP response element- (CRE) and AP-
1-directed transcription by CRE-transcription factor decoy oligonucleotide. gene-
specific inhibition of tumor growth. J Biol Chem, 1999. 274(3): p. 1573-80.
134. Ahn, J.D., et al., Inhibitory effects of novel AP-1 decoy oligodeoxynucleotides on
vascular smooth muscle cell proliferation in vitro and neointimal formation in
vivo. Circ Res, 2002. 90(12): p. 1325-32.
135. Stojanovic, T., et al., STAT-1 decoy oligonucleotide improves microcirculation
and reduces acute rejection in allogeneic rat small bowel transplants. Gene Ther,
2007. 14(11): p. 883-90.
136. Hashim, II, et al., Potential use of iontophoresis for transdermal delivery of NF-
kappaB decoy oligonucleotides. Int J Pharm, 2010. 393(1-2): p. 127-34.
137. Yasukawa, H., et al., Inhibition of intimal hyperplasia after balloon injury by
antibodies to intercellular adhesion molecule-1 and lymphocyte function-
associated antigen-1. Circulation, 1997. 95(6): p. 1515-22.
138. De Stefano, D., et al., Oligonucleotide decoy to NF-kappaB slowly released from
PLGA microspheres reduces chronic inflammation in rat. Pharmacol Res, 2009.
60(1): p. 33-40.
139. Nakamura, H., et al., Prevention and regression of atopic dermatitis by ointment
containing NF-kB decoy oligodeoxynucleotides in NC/Nga atopic mouse model.
Gene Ther, 2002. 9(18): p. 1221-9.
140. Desmet, C., et al., Selective blockade of NF-kappa B activity in airway immune
cells inhibits the effector phase of experimental asthma. J Immunol, 2004. 173(9):
p. 5766-75.
141. Tomita, T., et al., Suppressed severity of collagen-induced arthritis by in vivo
transfection of nuclear factor kappaB decoy oligodeoxynucleotides as a gene
therapy. Arthritis Rheum, 1999. 42(12): p. 2532-42.
142. Sakaue, G., et al., NF-kappa B decoy suppresses cytokine expression and thermal
hyperalgesia in a rat neuropathic pain model. Neuroreport, 2001. 12(10): p.
2079-84.
90
143. Ueno, T., et al., Nuclear factor-kappa B decoy attenuates neuronal damage after
global brain ischemia: a future strategy for brain protection during circulatory
arrest. J Thorac Cardiovasc Surg, 2001. 122(4): p. 720-7.
144. Wang, H., et al., Inhibition of tissue factor expression in brain microvascular
endothelial cells by nanoparticles loading NF-kappaB decoy oligonucleotides. Int
J Mol Sci, 2008. 9(9): p. 1851-62.
145. Jones, A.R. and E.V. Shusta, Blood-brain barrier transport of therapeutics via
receptor-mediation. Pharm Res, 2007. 24(9): p. 1759-71.
146. Descamps, L., et al., Receptor-mediated transcytosis of transferrin through blood-
brain barrier endothelial cells. Am J Physiol, 1996. 270(4 Pt 2): p. H1149-58.
147. Fishman, J.B., et al., Receptor-mediated transcytosis of transferrin across the
blood-brain barrier. J Neurosci Res, 1987. 18(2): p. 299-304.
148. Frank, H.J., et al., Enhanced insulin binding to blood-brain barrier in vivo and to
brain microvessels in vitro in newborn rabbits. Diabetes, 1985. 34(8): p. 728-33.
149. Pardridge, W.M., J. Eisenberg, and J. Yang, Human blood-brain barrier insulin
receptor. J Neurochem, 1985. 44(6): p. 1771-8.
150. Duffy, K.R., W.M. Pardridge, and R.G. Rosenfeld, Human blood-brain barrier
insulin-like growth factor receptor. Metabolism, 1988. 37(2): p. 136-40.
151. Demeule, M., et al., High transcytosis of melanotransferrin (P97) across the
blood-brain barrier. J Neurochem, 2002. 83(4): p. 924-33.
152. Gutierrez, E.G., W.A. Banks, and A.J. Kastin, Murine tumor necrosis factor
alpha is transported from blood to brain in the mouse. J Neuroimmunol, 1993.
47(2): p. 169-76.
153. Qian, Z.M., et al., Targeted drug delivery via the transferrin receptor-mediated
endocytosis pathway. Pharmacol Rev, 2002. 54(4): p. 561-87.
154. Malecki, E.A., et al., Existing and emerging mechanisms for transport of iron and
manganese to the brain. J Neurosci Res, 1999. 56(2): p. 113-22.
155. Moos, T. and E.H. Morgan, Transferrin and transferrin receptor function in brain
barrier systems. Cell Mol Neurobiol, 2000. 20(1): p. 77-95.
156. Mishra, V., et al., Targeted brain delivery of AZT via transferrin anchored
pegylated albumin nanoparticles. J Drug Target, 2006. 14(1): p. 45-53.
157. Zhou, Q.H., et al., Brain-penetrating tumor necrosis factor decoy receptor in the
mouse. Drug Metab Dispos, 2011. 39(1): p. 71-6.
158. Sumbria, R.K., R.J. Boado, and W.M. Pardridge, Brain protection from stroke
with intravenous TNFalpha decoy receptor-Trojan horse fusion protein. J Cereb
Blood Flow Metab, 2012. 32(10): p. 1933-8.
159. Zhou, Q.H., et al., Brain penetrating IgG-erythropoietin fusion protein is
neuroprotective following intravenous treatment in Parkinson's disease in the
mouse. Brain Res, 2011. 1382: p. 315-20.
160. Li, X., Q. Zhao, and L. Qiu, Smart ligand: aptamer-mediated targeted delivery of
chemotherapeutic drugs and siRNA for cancer therapy. J Control Release, 2013.
171(2): p. 152-62.
161. Que-Gewirth, N.S. and B.A. Sullenger, Gene therapy progress and prospects:
RNA aptamers. Gene Ther, 2007. 14(4): p. 283-91.
162. Sudimack, J. and R.J. Lee, Targeted drug delivery via the folate receptor. Adv
Drug Deliv Rev, 2000. 41(2): p. 147-62.
91
163. Lu, Y. and P.S. Low, Folate-mediated delivery of macromolecular anticancer
therapeutic agents. Adv Drug Deliv Rev, 2002. 54(5): p. 675-93.
164. Bruno, J.G., A review of therapeutic aptamer conjugates with emphasis on new
approaches. Pharmaceuticals (Basel), 2013. 6(3): p. 340-57.
165. Jin, E., et al., Acid-active cell-penetrating peptides for in vivo tumor-targeted
drug delivery. J Am Chem Soc, 2013. 135(2): p. 933-40.
166. Sun, H., et al., Oligonucleotide aptamers: new tools for targeted cancer therapy.
Mol Ther Nucleic Acids, 2014. 3: p. e182.
167. Eyetech Study, G., Preclinical and phase 1A clinical evaluation of an anti-VEGF
pegylated aptamer (EYE001) for the treatment of exudative age-related macular
degeneration. Retina, 2002. 22(2): p. 143-52.
168. Chu, T.C., et al., Aptamer mediated siRNA delivery. Nucleic Acids Res, 2006.
34(10): p. e73.
169. Dassie, J.P., et al., Systemic administration of optimized aptamer-siRNA chimeras
promotes regression of PSMA-expressing tumors. Nat Biotechnol, 2009. 27(9): p.
839-49.
170. Ni, X., et al., Prostate-targeted radiosensitization via aptamer-shRNA chimeras in
human tumor xenografts. J Clin Invest, 2011. 121(6): p. 2383-90.
171. Boyacioglu, O., et al., Dimeric DNA Aptamer Complexes for High-capacity-
targeted Drug Delivery Using pH-sensitive Covalent Linkages. Mol Ther Nucleic
Acids, 2013. 2: p. e107.
172. Bagalkot, V., et al., An aptamer-doxorubicin physical conjugate as a novel
targeted drug-delivery platform. Angew Chem Int Ed Engl, 2006. 45(48): p.
8149-52.
173. Subramanian, N., et al., Target-specific delivery of doxorubicin to retinoblastoma
using epithelial cell adhesion molecule aptamer. Mol Vis, 2012. 18: p. 2783-95.
174. Shieh, Y.A., et al., Aptamer-based tumor-targeted drug delivery for
photodynamic therapy. ACS Nano, 2010. 4(3): p. 1433-42.
175. Wu, J., et al., Nucleolin targeting AS1411 modified protein nanoparticle for
antitumor drugs delivery. Mol Pharm, 2013. 10(10): p. 3555-63.
176. Hu, Y., et al., Novel MUC1 aptamer selectively delivers cytotoxic agent to cancer
cells in vitro. PLoS One, 2012. 7(2): p. e31970.
177. Thiel, K.W., et al., Delivery of chemo-sensitizing siRNAs to HER2+-breast
cancer cells using RNA aptamers. Nucleic Acids Res, 2012. 40(13): p. 6319-37.
178. Liu, Z., et al., Novel HER2 aptamer selectively delivers cytotoxic drug to HER2-
positive breast cancer cells in vitro. J Transl Med, 2012. 10: p. 148.
179. Zhou, J., et al., Novel dual inhibitory function aptamer-siRNA delivery system for
HIV-1 therapy. Mol Ther, 2008. 16(8): p. 1481-9.
180. Zhou, J., et al., Functional in vivo delivery of multiplexed anti-HIV-1 siRNAs via a
chemically synthesized aptamer with a sticky bridge. Mol Ther, 2013. 21(1): p.
192-200.
181. Hu, J., et al., Inhibition of monocyte adhesion to brain-derived endothelial cells
by dual functional RNA chimeras. Mol Ther Nucleic Acids, 2014. 3: p. e209.
182. Bates, P.J., et al., Discovery and development of the G-rich oligonucleotide
AS1411 as a novel treatment for cancer. Exp Mol Pathol, 2009. 86(3): p. 151-64.
92
183. Mu, C., et al., Solubilization of flurbiprofen into aptamer-modified PEG-PLA
micelles for targeted delivery to brain-derived endothelial cells in vitro. J
Microencapsul, 2013. 30(7): p. 701-8.
184. Kang, Y.S., R.J. Boado, and W.M. Pardridge, Pharmacokinetics and organ
clearance of a 3'-biotinylated, internally [32P]-labeled phosphodiester
oligodeoxynucleotide coupled to a neutral avidin/monoclonal antibody conjugate.
Drug Metab Dispos, 1995. 23(1): p. 55-9.
185. Hu, J., C. Mu, and J. Hao, Cerebral ischemia reduces expression of Hs1-
associated protein X-1 (Hax-1) in mouse brain. Neurosci Lett, 2013. 534: p. 338-
43.
186. Solan, N.J., et al., RelB cellular regulation and transcriptional activity are
regulated by p100. J Biol Chem, 2002. 277(2): p. 1405-18.
187. Kunath, K., et al., The structure of PEG-modified poly(ethylene imines) influences
biodistribution and pharmacokinetics of their complexes with NF-kappaB decoy
in mice. Pharm Res, 2002. 19(6): p. 810-7.
188. Wu, Z., F.M. Hofman, and B.V. Zlokovic, A simple method for isolation and
characterization of mouse brain microvascular endothelial cells. J Neurosci
Methods, 2003. 130(1): p. 53-63.
189. Spulber, S., et al., Molecular hydrogen reduces LPS-induced neuroinflammation
and promotes recovery from sickness behaviour in mice. PLoS One, 2012. 7(7): p.
e42078.
190. Memet, S., NF-kappaB functions in the nervous system: from development to
disease. Biochem Pharmacol, 2006. 72(9): p. 1180-95.
191. Meffert, M.K. and D. Baltimore, Physiological functions for brain NF-kappaB.
Trends Neurosci, 2005. 28(1): p. 37-43.
192. Mattson, M.P. and M.K. Meffert, Roles for NF-kappaB in nerve cell survival,
plasticity, and disease. Cell Death Differ, 2006. 13(5): p. 852-60.
193. Schneider, A., et al., NF-kappaB is activated and promotes cell death in focal
cerebral ischemia. Nat Med, 1999. 5(5): p. 554-9.
194. Nurmi, A., et al., Nuclear factor-kappaB contributes to infarction after permanent
focal ischemia. Stroke, 2004. 35(4): p. 987-91.
195. Xu, L., et al., Recombinant adenoviral expression of dominant negative
IkappaBalpha protects brain from cerebral ischemic injury. Biochem Biophys
Res Commun, 2002. 299(1): p. 14-7.
196. Shen, W.H., C.Y. Zhang, and G.Y. Zhang, Antioxidants attenuate reperfusion
injury after global brain ischemia through inhibiting nuclear factor-kappa B
activity in rats. Acta Pharmacol Sin, 2003. 24(11): p. 1125-30.
197. Desai, A., N. Singh, and R. Raghubir, Neuroprotective potential of the NF-
kappaB inhibitor peptide IKK-NBD in cerebral ischemia-reperfusion injury.
Neurochem Int, 2010. 57(8): p. 876-83.
198. Duckworth, E.A., et al., NF-kappaB protects neurons from ischemic injury after
middle cerebral artery occlusion in mice. Brain Res, 2006. 1088(1): p. 167-75.
199. Blondeau, N., et al., Activation of the nuclear factor-kappaB is a key event in
brain tolerance. J Neurosci, 2001. 21(13): p. 4668-77.
200. Nijboer, C.H., et al., A dual role of the NF-kappaB pathway in neonatal hypoxic-
ischemic brain damage. Stroke, 2008. 39(9): p. 2578-86.
93
201. Bhakar, A.L., et al., Constitutive nuclear factor-kappa B activity is required for
central neuron survival. J Neurosci, 2002. 22(19): p. 8466-75.
202. Emmanouil, M., et al., Neuronal I kappa B kinase beta protects mice from
autoimmune encephalomyelitis by mediating neuroprotective and
immunosuppressive effects in the central nervous system. J Immunol, 2009.
183(12): p. 7877-89.
203. Brambilla, R., et al., Transgenic inhibition of astroglial NF-kappa B improves
functional outcome in experimental autoimmune encephalomyelitis by
suppressing chronic central nervous system inflammation. J Immunol, 2009.
182(5): p. 2628-40.
204. Cho, I.H., et al., Role of microglial IKKbeta in kainic acid-induced hippocampal
neuronal cell death. Brain, 2008. 131(Pt 11): p. 3019-33.
205. Yang, T., K.E. Roder, and T.J. Abbruscato, Evaluation of bEnd5 cell line as an in
vitro model for the blood-brain barrier under normal and hypoxic/aglycemic
conditions. J Pharm Sci, 2007. 96(12): p. 3196-213.
206. Omori, N., et al., Targeting of post-ischemic cerebral endothelium in rat by
liposomes bearing polyethylene glycol-coupled transferrin. Neurol Res, 2003.
25(3): p. 275-9.
207. Tuma, P. and A.L. Hubbard, Transcytosis: crossing cellular barriers. Physiol
Rev, 2003. 83(3): p. 871-932.
208. Moos, T., Age-dependent uptake and retrograde axonal transport of exogenous
albumin and transferrin in rat motor neurons. Brain Res, 1995. 672(1-2): p. 14-
23.
209. Giometto, B., et al., Transferrin receptors in rat central nervous system. An
immunocytochemical study. J Neurol Sci, 1990. 98(1): p. 81-90.
210. Orita, T., et al., Transferrin receptors in injured brain. Acta Neuropathol, 1990.
79(6): p. 686-8.
211. Dufes, C., M. Al Robaian, and S. Somani, Transferrin and the transferrin
receptor for the targeted delivery of therapeutic agents to the brain and cancer
cells. Ther Deliv, 2013. 4(5): p. 629-40.
212. Yu, Y.J. and R.J. Watts, Developing therapeutic antibodies for neurodegenerative
disease. Neurotherapeutics, 2013. 10(3): p. 459-72.
213. Bien-Ly, N., et al., Transferrin receptor (TfR) trafficking determines brain uptake
of TfR antibody affinity variants. J Exp Med, 2014. 211(2): p. 233-44.
214. Moos, T. and E.H. Morgan, Restricted transport of anti-transferrin receptor
antibody (OX26) through the blood-brain barrier in the rat. J Neurochem, 2001.
79(1): p. 119-29.
215. Paris-Robidas, S., et al., In vivo labeling of brain capillary endothelial cells after
intravenous injection of monoclonal antibodies targeting the transferrin receptor.
Mol Pharmacol, 2011. 80(1): p. 32-9.
216. Corinne Benakis LH, R.A.D.P., inflammation and stroke. kardiovaskulare
Medizin, 2008. 12(5): p. 143-150.
217. Tomita, T., et al., Transcription factor decoy for NFkappaB inhibits cytokine and
adhesion molecule expressions in synovial cells derived from rheumatoid
arthritis. Rheumatology (Oxford), 2000. 39(7): p. 749-57.
94
218. Zhou, J., et al., Selection, characterization and application of new RNA HIV gp
120 aptamers for facile delivery of Dicer substrate siRNAs into HIV infected cells.
Nucleic Acids Res, 2009. 37(9): p. 3094-109.
219. Griesenbach, U., et al., Anti-inflammatory gene therapy directed at the airway
epithelium. Gene Ther, 2000. 7(4): p. 306-13.
220. Griesenbach, U., et al., Cytoplasmic deposition of NFkappaB decoy
oligonucleotides is insufficient to inhibit bleomycin-induced pulmonary
inflammation. Gene Ther, 2002. 9(16): p. 1109-15.
221. Liu, Y., et al., [Nuclear localization of oligonucleotides decoy effect on nuclear
factor-kappaB activity]. Sheng Wu Gong Cheng Xue Bao, 2010. 26(12): p. 1683-
9.
222. Bene, A., R.C. Kurten, and T.C. Chambers, Subcellular localization as a limiting
factor for utilization of decoy oligonucleotides. Nucleic Acids Res, 2004. 32(19):
p. e142.
223. Abdelmawla, S., et al., Pharmacological characterization of chemically
synthesized monomeric phi29 pRNA nanoparticles for systemic delivery. Mol
Ther, 2011. 19(7): p. 1312-22.
224. Healy, J.M., et al., Pharmacokinetics and biodistribution of novel aptamer
compositions. Pharm Res, 2004. 21(12): p. 2234-46.
225. Whiteside, C. and M. Silverman, Determination of postglomerular permselectivity
to neutral dextrans in the dog. Am J Physiol, 1983. 245(4): p. F496-505.
226. Geary, R.S., et al., Pharmacokinetic properties of 2'-O-(2-methoxyethyl)-modified
oligonucleotide analogs in rats. J Pharmacol Exp Ther, 2001. 296(3): p. 890-7.
227. Crooke, S.T., et al., Pharmacokinetic properties of several novel oligonucleotide
analogs in mice. J Pharmacol Exp Ther, 1996. 277(2): p. 923-37.
228. PRaR, R.W., Aptamers for targeted drug delivery. Pharmaceuticals (Basel), 2010.
3(18).
229. Pardridge, W.M., Blood-brain barrier drug delivery of IgG fusion proteins with a
transferrin receptor monoclonal antibody. Expert Opin Drug Deliv, 2015. 12(2):
p. 207-22.
230. Pardridge, W.M., Brain drug targeting and gene technologies. Jpn J Pharmacol,
2001. 87(2): p. 97-103.
231. Song, B.W., et al., Enhanced neuroprotective effects of basic fibroblast growth
factor in regional brain ischemia after conjugation to a blood-brain barrier
delivery vector. J Pharmacol Exp Ther, 2002. 301(2): p. 605-10.
232. Pang, Z., et al., Preparation and brain delivery property of biodegradable
polymersomes conjugated with OX26. J Control Release, 2008. 128(2): p. 120-7.
233. Park, S. and P.J. Sinko, P-glycoprotein and mutlidrug resistance-associated
proteins limit the brain uptake of saquinavir in mice. J Pharmacol Exp Ther,
2005. 312(3): p. 1249-56.
234. Wu, D., et al., Blood-brain barrier permeability to morphine-6-glucuronide is
markedly reduced compared with morphine. Drug Metab Dispos, 1997. 25(6): p.
768-71.
235. Xie, R., et al., The role of P-glycoprotein in blood-brain barrier transport of
morphine: transcortical microdialysis studies in mdr1a (-/-) and mdr1a (+/+)
mice. Br J Pharmacol, 1999. 128(3): p. 563-8.
95
236. Letrent, S.P., et al., Effect of GF120918, a potent P-glycoprotein inhibitor, on
morphine pharmacokinetics and pharmacodynamics in the rat. Pharm Res, 1998.
15(4): p. 599-605.
237. Letrent, S.P., et al., Effects of a potent and specific P-glycoprotein inhibitor on the
blood-brain barrier distribution and antinociceptive effect of morphine in the rat.
Drug Metab Dispos, 1999. 27(7): p. 827-34.
238. Lee, H.J., et al., Targeting rat anti-mouse transferrin receptor monoclonal
antibodies through blood-brain barrier in mouse. J Pharmacol Exp Ther, 2000.
292(3): p. 1048-52.
239. Herber, D.L., et al., Time-dependent reduction in Abeta levels after intracranial
LPS administration in APP transgenic mice. Exp Neurol, 2004. 190(1): p. 245-53.
240. Zhang, G. and S. Ghosh, Molecular mechanisms of NF-kappaB activation
induced by bacterial lipopolysaccharide through Toll-like receptors. J Endotoxin
Res, 2000. 6(6): p. 453-7.
241. Luheshi, G.N., Cytokines and fever. Mechanisms and sites of action. Ann N Y
Acad Sci, 1998. 856: p. 83-9.
242. Kilbourn, R.G. and P. Belloni, Endothelial cell production of nitrogen oxides in
response to interferon gamma in combination with tumor necrosis factor,
interleukin-1, or endotoxin. J Natl Cancer Inst, 1990. 82(9): p. 772-6.
243. Rivest, S., Molecular insights on the cerebral innate immune system. Brain Behav
Immun, 2003. 17(1): p. 13-9.
244. Matsumura, K., et al., Cyclooxygenase in the vagal afferents: is it involved in the
brain prostaglandin response evoked by lipopolysaccharide? Auton Neurosci,
2000. 85(1-3): p. 88-92.
245. Cao, C., et al., Induction by lipopolysaccharide of cyclooxygenase-2 mRNA in rat
brain; its possible role in the febrile response. Brain Res, 1995. 697(1-2): p. 187-
96.
246. Dantzer, R. and K.W. Kelley, Twenty years of research on cytokine-induced
sickness behavior. Brain Behav Immun, 2007. 21(2): p. 153-60.
247. Yang, F., et al., IKK beta plays an essential role in the phosphorylation of
RelA/p65 on serine 536 induced by lipopolysaccharide. J Immunol, 2003.
170(11): p. 5630-5.
248. !!! INVALID CITATION !!!
249. Higuchi, Y., S. Kawakami, and M. Hashida, Strategies for in vivo delivery of
siRNAs: recent progress. BioDrugs, 2010. 24(3): p. 195-205.
250. King, D.J., et al., Novel combinatorial selection of phosphorothioate
oligonucleotide aptamers. Biochemistry, 1998. 37(47): p. 16489-93.
251. Wang, R.E., et al., Improving the stability of aptamers by chemical modification.
Curr Med Chem, 2011. 18(27): p. 4126-38.
252. Pozmogova, G.E., et al., Anticoagulant effects of thioanalogs of thrombin-binding
DNA-aptamer and their stability in the plasma. Bull Exp Biol Med, 2010. 150(2):
p. 180-4.
253. Dougan, H., et al., Extending the lifetime of anticoagulant oligodeoxynucleotide
aptamers in blood. Nucl Med Biol, 2000. 27(3): p. 289-97.
254. Shaw, J.P., et al., Modified deoxyoligonucleotides stable to exonuclease
degradation in serum. Nucleic Acids Res, 1991. 19(4): p. 747-50.
96
255. Shum, K.T. and J.A. Tanner, Differential inhibitory activities and stabilisation of
DNA aptamers against the SARS coronavirus helicase. Chembiochem, 2008.
9(18): p. 3037-45.
256. Boomer, R.M., et al., Conjugation to polyethylene glycol polymer promotes
aptamer biodistribution to healthy and inflamed tissues. Oligonucleotides, 2005.
15(3): p. 183-95.
257. Da Pieve, C., et al., PEGylation and biodistribution of an anti-MUC1 aptamer in
MCF-7 tumor-bearing mice. Bioconjug Chem, 2012. 23(7): p. 1377-81.