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. 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
ii 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.
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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.
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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.
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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
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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
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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
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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,
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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
xi 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
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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
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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
1 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-
2
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)
3 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
4 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.
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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].
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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
7 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
8 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].
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(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
10 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
11 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
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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
13 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
14
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
15 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,
16
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
17 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.
18
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].
19
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
20 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
21
[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],
22
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
23 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.
24
(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-
25 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].
26
(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
27 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.
28
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.
29
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.
30
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.
31
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
32
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
33 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
34 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
35 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-
36 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
37 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
38 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.
39
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.
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).
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
41
(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
42
(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
43 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.
44
(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
45 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）
.
46
(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)
47
(b)
30 ##
20
* 10
VCAM-1 mRNA expressionVCAM-1 mRNA
(normalized to media control)) 0
B TNF-
+GS24-NF media control
+Scramble control TNF-
TNF-
(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.
48
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)
49
(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.
50
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.
51
(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)).
52
(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)).
53
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.
54
100 GS24-NFB TCA-precipitable GS24-NFB
10
1
0.1
Plasma concentraion( %ID/ml) concentraion( Plasma 0 20 40 60 80 Time(min)
(a)
100 TCA-precipitable ODN TCA-precipitable scramble ODN
10
1
0.1
Plasma concentraion( %ID/ml) concentraion( Plasma 0 20 40 60 80 Time(min)
(b)
Figure 20 Plasma concentration time profiles: (a) GS24-NFκB and TCA-precipitable GS24-NFκB; (b) GS24-NFκB and scramble GS24-NFκB
55
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.
56
16 14 12 10 8 6 4
Organdistribution (%ID/g) 2
0
sample sample sample sample sample sample
scramble scramble scramble scramble scramble scramble
heart lung liver spleen kidney brain
(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
57 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).
58
(a)
250 ###
200 ** 150
100
p-p65 expression p-p65 50
(% of media control) (% media of 0
B B
media control LPS+GS24-NF
LPS+scramble GS24-NF
(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.
59
(a)
250 ###
200 * 150
100
50
VCAM-1 expression VCAM-1
(% of media control) (% media of 0
B B
media control LPS+GS24-NF
LPS+scramble GS24-NF
(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.
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
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
62 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-
63 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
64 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
65 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-
66
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
67 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
68 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
69 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
70 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
71 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
72 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
73
[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
74
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
75 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,
76
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.
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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
78 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
79 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.
80
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.
81
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