JGV Papers in Press. Published May 14, 2014 as doi:10.1099/vir.0.066084-0
1
Delivery of anti-viral siRNA with gold-nanoparticles inhibits dengue virus infection in vitro
2 3
Running title: AuNPs deliver anti-dengue siRNA
4 5
Amber M. Paul1, Yongliang Shi2, Dhiraj Acharya1, Jessica R. Douglas1, Amanda Cooley1, John
6
F. Anderson3, Faqing Huang2 and Fengwei Bai1*
7 8 9
1
Department of Biological Sciences, 2Department of Chemistry and Biochemistry, University of
Southern Mississippi, Hattiesburg, MS 39406, USA.
10
3
11
06504, USA
Department of Entomology, Connecticut Agricultural Experiment Station, New Haven, CT
12 13 14 15 16
Word count (Summary): 162 words
17
Word count (Main Text): 4330 words
18
Journal’s Content’s Category: Human and Animal virus- Positive-stranded RNA
19
Number of figure: 5 and number of table: 0.
20 21 *
Corresponding Author: Fengwei Bai, Ph.D., Department of Biological Sciences, The University of Southern Mississippi, 118 College Drive # 5018, Hattiesburg, MS 39406, USA. Email: [email protected]; Telephone 601-266-4748; Fax: 601-266-5797
AuNPs deliver anti-Dengue siRNA
22
Summary
23
Dengue virus (DENV) infection in humans can cause flu-like illness, life threatening
24
hemorrhagic fever, or even death. There is no specific anti-DENV therapeutic or approved
25
vaccine currently available, partially due to the possibility of antibody-dependent enhancement
26
reaction. Small interfering RNAs (siRNAs) that target specific viral genes are considered a
27
promising therapeutic alternative against DENV infection. However, in vivo, siRNAs are
28
vulnerable to degradation by serum-nucleases and rapid renal excretion due to their small size
29
and anionic character. To enhance siRNA delivery and stability, we complexed anti-DENV
30
siRNAs with biocompatible gold nanoparticles (AuNPs) and tested them in vitro. We found that
31
cationic AuNP-siRNA complexes can enter Vero cells and significantly reduce DENV-serotype
32
2 replication and infectious virion release at both pre- and post-infection conditions. In addition,
33
RNase-treated AuNP-siRNA complexes can still inhibit DENV replication, suggesting that
34
AuNPs maintain siRNA stability. Collectively, these results demonstrate that AuNPs are able to
35
efficiently deliver siRNAs and control infection in vitro, indicating a novel anti-DENV strategy.
36 37 38
Keywords: dengue virus, siRNA delivery, gold nanoparticle
AuNPs deliver anti-Dengue siRNA
39
Introduction
40
Dengue virus (DENV) is a single-stranded, positive-sense RNA virus belonging to the
41
family Flaviviridae. DENV is transmitted to humans primarily by the mosquito vectors, Aedes
42
aegypti and Aedes albopictus (Xi et al., 2008). After DENV binds to host cellular receptors, it is
43
endocytosed and uncoated within the endosome to release its RNA genome into the cytoplasm,
44
where replication of the viral genome and assembly of virions occur (van der Schaar et al.,
45
2008). The common symptoms of DENV infection in humans are characterized by fever,
46
headache, joint and muscle pain and measle-like rashes (Halstead, 2007; Wilder-Smith et al.,
47
2010). Although asymptomatic in most cases, DENV infection can lead to serious disease, such
48
as hemorrhagic fever and systemic shock syndrome that can potentially be fatal. There are four
49
serotypes of DENV (DENV-1, DENV-2, DENV-3 and DENV-4) and illness can be produced by
50
any serotype (Tomashek & Margolis, 2011). Although recovery from infection to one serotype
51
provides lifelong immunity against that particular serotype, subsequent infection by other co-
52
circulating serotypes may cause an antibody-dependent enhancement reaction, which increases
53
the risk of developing severe dengue hemorrhagic fever, shock and even death (Halstead, 1982;
54
Kliks et al., 1989). DENV infection has been reported in more than 100 countries, including the
55
United States (Gubler, 2002; Ramos et al., 2008; Tomashek & Margolis, 2011). A recent report
56
has estimated that 390 million people are infected with DENV worldwide, with 96 million
57
people manifesting clinical symptoms of disease (Bhatt et al., 2013).
58
Since antibody-dependent enhancement hinders the development of an antibody-based
59
vaccine, the development of therapeutics that target replication or expression of the viral genome
60
may serve as promising alternatives to control DENV infection. Anti-host receptor (Alhoot et al.,
61
2011) and anti-DENV (Stein et al., 2011; Subramanya et al., 2010) small interfering RNAs
3
AuNPs deliver anti-Dengue siRNA
62
(siRNAs) have considerably advanced both clinical- and laboratory-based applications as
63
potential DENV therapeutic alternatives (Idrees et al., 2013). However, efficient delivery of
64
siRNAs remains a great challenge for clinical applications. The most common siRNA delivery
65
approach is with a lipid-based transfection reagent, whereby the negatively charged siRNA
66
duplex binds to the cationic liposome transfection reagent, which allows for cell membrane
67
endocytosis, micelle formation and intracellular release of the siRNA into the cytosol (Zuhorn et
68
al., 2002). Although this method is successful for siRNA delivery in vitro, it is not an ideal
69
candidate to use in vivo, as these chemically-based transfection reagents are physiologically toxic
70
and could induce cellular stress or aberrant transcriptional profiles (Torchilin, 2005; Zhong et al.,
71
2008).
72
Recently, gold nanoparticles (AuNPs) have been suggested as promising vehicles to
73
deliver siRNA both in vitro and in vivo (Conde et al., 2012; Conde et al., 2013; Mitra et al.,
74
2013; Yang et al., 2013). Several advantages make them well suited for safe and efficient
75
protection and transport of siRNA: a) biocompatibility (Chithrani & Chan, 2007; Lewinski et
76
al., 2008); b) ease of preparation and facile surface modification (Elbakry et al., 2009; Lee et al.,
77
2011; Song et al., 2010); and c) high siRNA loading capacity. In this study, we constructed novel
78
anti-DENV AuNP-siRNA nanoplexes and found that AuNPs are able to promote siRNA stability
79
and efficiently deliver anti-DENV siRNAs in vitro.
80
4
AuNPs deliver anti-Dengue siRNA
81
Results
82
Antiviral siRNAs efficiently inhibit DENV replication
83
Two antiviral siRNAs (si-1 and si-3) were designed to target the most prevalent dengue
84
serotype, DENV-2. Vero cells were transfected with siRNA (20 nM) using a lipofectamine-based
85
transfection reagent (TR) and subsequently infected with DENV-2 (multiplicity of infection,
86
MOI = 0.1). Sequence scrambled siRNA (siSCRM) was used as a negative control and a
87
previously described anti-DENV siRNA (DC-3) was used as a positive control (Stein et al.,
88
2011). At 48 hr post-infection, the cells were harvested for total RNA extraction, cDNA
89
synthesis and subjected to real-time quantitative polymerase chain reaction (Q-PCR) for the
90
measurement of DENV capsid gene and cellular β-actin gene expression. The Q-PCR results
91
showed that si-1, si-3 and DC-3 significantly reduced DENV-2 capsid gene expression compared
92
to the control, in which cells were treated with TR only (p < 0.001, Fig. 1a). In contrast, siSCRM
93
did not show any inhibitory effect (p = ns, Fig. 1a), suggesting that siRNA inhibition is
94
sequence-specific. To test if infectious viral particles released from the infected cells were
95
reduced with siRNA transfection, the media were quantified by a plaque forming assay (plaque
96
forming unit, PFU) and an immunostaining assay (focus forming unit, FFU), with the latter
97
detecting viral envelope (E) protein expression. Since the DENV-2 genome shares some
98
homology with other DENV-serotypes, the antiviral effect of si-1 and si-3 (20 nM) was also
99
evaluated against the replication of DENV-1, DENV-3 and DENV-4 (MOI = 0.1). Q-PCR
100
analysis showed that si-1 and si-3 were able to inhibit DENV-3 and DENV-4 replication, but
101
showed no inhibitory activity for DENV-1, compared to the control in which cells were treated
102
with TR, without siRNA (p < 0.001 and p = ns, Fig. 1d-f). RNA sequence alignment analysis
103
suggested that the lack of inhibition of si-1 and si-3 against DENV-1 replication might be due to
5
AuNPs deliver anti-Dengue siRNA
104
lower homology between their targeting sequences (approximately 50% and 70% respectively,
105
data not shown). In summary, these results demonstrated that both si-1 and si-3 were able to
106
significantly inhibit replication of DENV serotypes 2, 3 and 4.
107 108
Design and optimization of AuNP complexes for anti-DENV siRNA delivery in vitro
109
Recently, AuNPs have been designed and optimized as delivery vehicles for siRNA both
110
in vitro and in vivo (Conde et al., 2013; Gilleron et al., 2013; Mitra et al., 2013; Yang et al.,
111
2013; Zheng et al., 2012). Optimization of AuNPs has been focused on certain properties that
112
enhance siRNA delivery and reduce possible cytotoxicity. Such properties include surface
113
charge, surface complexity and size of the AuNPs (De Jong & Borm, 2008). In this study, three
114
AuNP-siRNA complexes (anionic, neutral and cationic) were prepared by a novel layer-by-layer
115
(LbL) approach to deliver anti-DENV siRNA into Vero cells. To confirm each layer was
116
successfully coated on the AuNPs, the AuNP complexes were characterized by UV-Vis spectra,
117
DLS and Zeta potential. AuNPs and AuNP-PEI-siRNAs (PEI: polyethylenimine) were measured
118
by UV-Vis, which disclosed a shift in the Surface Plasmon Resonance (SPR) peaks with the
119
addition of siRNA (Fig. 2a). DLS measured the hydrodynamic diameters of the different
120
complexes: AuNPs (12.92 nm), AuNP-PEI (24.97 nm), AuNP-PEI-siRNA (41.79 nm), and
121
AuNP-PEI-siRNA-PEI (43.25 nm), which indicated that the diameters increased with the
122
addition of each layer (Fig. 2b). AuNPs were first coated with an inner layer of HS-PEI (thiol-
123
labeled PEI) via Au-S bonds, rendering the particles cationic. The negatively charged siRNAs
124
were then coated on the AuNP-PEIs through electrostatic interactions, rendering the complexes
125
anionic. Adjusting the concentration of the outer layer PEI with the AuNP-PEI-siRNA
126
complexes (Fig. 2c) altered the overall charge from negative to positive. Zeta potential
6
AuNPs deliver anti-Dengue siRNA
127
determined the surface charges of the complexes: AuNPs (-34.9 mV), AuNP-PEI (46.9 mV),
128
AuNP-PEI-siRNA (-38.8 mV) and AuNP-PEI-siRNA-PEI (53.3 mV) (Fig. 2d, neutral charge
129
data not shown). These results demonstrated the optimization of AuNPs and successful synthesis
130
of AuNP-siRNA complexes.
131
To test the effect of surface charge on siRNA delivery efficiency, we treated Vero cells
132
with anionic (-40 mV), neutral (0 mV) and cationic (+40 mV) AuNP-siRNA complexes (20 nM)
133
for 48 hrs and subsequently infected them with DENV-2 (MOI = 0.1) for an additional 48 hrs. Q-
134
PCR analysis showed that cationic AuNP-si-1 and AuNP-si-3 complexes significantly reduced
135
DENV-2 replication (p < 0.005, Fig. 2e). While anionic AuNP-si-3, but not AuNP-si-1
136
complexes, also inhibited DENV-2 replication compared to the control, in which cells were
137
infected with DENV-2 only (p < 0.05, Fig. 2e). In contrast, neither the neutrally charged AuNP-
138
si-1 nor the AuNP-si-3 complex inhibited viral replication, suggesting inefficient delivery of
139
siRNAs by neutral AuNP-siRNA complexes (Fig. 2e). In summary, these results showed that
140
cationic AuNP-siRNA complexes could effectively deliver anti-DENV siRNAs into cells.
141
To confirm cationic AuNP-siRNAs are able to enter cells, Vero cells were cultured with
142
cationic AuNP-si-1 (80nM) for 48 hrs and subsequently infected with DENV-2 (MOI = 0.1) for
143
an additional 72 hrs. Ultra-thin sections embedded in epoxy resin were imaged using a
144
transmission electron microscope (TEM). The TEM images showed that AuNPs localized within
145
the cytoplasm of Vero cells (Fig. 2f, g and inserts), which implied AuNP complexes were able to
146
deliver siRNAs into the cells.
147 148
Cationic AuNP-siRNA complexes are non-toxic to Vero cells
7
AuNPs deliver anti-Dengue siRNA
149
AuNPs have been noted as biocompatible vehicles for drug delivery (Connor et al., 2005;
150
Shukla et al., 2005). However, they also have been suggested to have some cytotoxicity due to
151
their cationic polarity and surface modifications (Gerber et al., 2013; Goodman et al., 2004). To
152
exclude the possibility that inhibition of DENV-2 replication was due to the AuNP-siRNA
153
complexes causing Vero cell death, we assessed the cytotoxicity of the AuNP-siRNA complexes
154
when exposed to Vero cells. We incubated AuNP-siRNA complexes (+40 mV) with Vero cells
155
at the same experimental conditions and concentrations that were used for the pre-treatment
156
transfection assays, without DENV-2 infection. Cellular morphology was imaged and cell
157
number was quantified by using DAPI-nuclear staining and examined under a confocal
158
microscope. No morphological changes (Fig. 3a, i and ii) or cell number reduction (Fig. 3b) were
159
observed with any AuNP-siRNA treated samples compared to the untreated control. To further
160
confirm this, cellular lactate dehydrogenase (LDH) released from broken cell membrane was
161
also measured. The LDH cytotoxicity assay results showed no significant toxicity (≤ 6.5%
162
cytotoxicity), which was similar with the untreated cell control (2% cytotoxicity, Fig. 3c). These
163
data suggested that Vero cells exposed to AuNP-siRNA complexes have negligible cytotoxicity,
164
which indicated that inhibition of DENV-2 replication was due to siRNA mediated RNA
165
interference.
166 167
Cationic AuNP-siRNA complexes inhibit DENV-2 infection in both pre- and post-infection
168
conditions
169
To further confirm that cationic AuNP-siRNA complexes inhibit DENV-2 replication, we
170
pre-treated Vero cells with cationic AuNP-siRNAs (+40 mV) at 20 nM and 80 nM for 48 hrs and
171
subsequently infected them with DENV-2 (MOI = 0.1) for an additional 48 and 72 hrs, followed
8
AuNPs deliver anti-Dengue siRNA
172
by Q-PCR analysis. The results showed that compared to DENV-2 only infected controls,
173
AuNP-si-1 (20 nM) reduced DENV replication approximately 2-fold at both 48 and 72 hrs (p
1
Delivery of anti-viral siRNA with gold-nanoparticles inhibits dengue virus infection in vitro
2 3
Running title: AuNPs deliver anti-dengue siRNA
4 5
Amber M. Paul1, Yongliang Shi2, Dhiraj Acharya1, Jessica R. Douglas1, Amanda Cooley1, John
6
F. Anderson3, Faqing Huang2 and Fengwei Bai1*
7 8 9
1
Department of Biological Sciences, 2Department of Chemistry and Biochemistry, University of
Southern Mississippi, Hattiesburg, MS 39406, USA.
10
3
11
06504, USA
Department of Entomology, Connecticut Agricultural Experiment Station, New Haven, CT
12 13 14 15 16
Word count (Summary): 162 words
17
Word count (Main Text): 4330 words
18
Journal’s Content’s Category: Human and Animal virus- Positive-stranded RNA
19
Number of figure: 5 and number of table: 0.
20 21 *
Corresponding Author: Fengwei Bai, Ph.D., Department of Biological Sciences, The University of Southern Mississippi, 118 College Drive # 5018, Hattiesburg, MS 39406, USA. Email: [email protected]; Telephone 601-266-4748; Fax: 601-266-5797
AuNPs deliver anti-Dengue siRNA
22
Summary
23
Dengue virus (DENV) infection in humans can cause flu-like illness, life threatening
24
hemorrhagic fever, or even death. There is no specific anti-DENV therapeutic or approved
25
vaccine currently available, partially due to the possibility of antibody-dependent enhancement
26
reaction. Small interfering RNAs (siRNAs) that target specific viral genes are considered a
27
promising therapeutic alternative against DENV infection. However, in vivo, siRNAs are
28
vulnerable to degradation by serum-nucleases and rapid renal excretion due to their small size
29
and anionic character. To enhance siRNA delivery and stability, we complexed anti-DENV
30
siRNAs with biocompatible gold nanoparticles (AuNPs) and tested them in vitro. We found that
31
cationic AuNP-siRNA complexes can enter Vero cells and significantly reduce DENV-serotype
32
2 replication and infectious virion release at both pre- and post-infection conditions. In addition,
33
RNase-treated AuNP-siRNA complexes can still inhibit DENV replication, suggesting that
34
AuNPs maintain siRNA stability. Collectively, these results demonstrate that AuNPs are able to
35
efficiently deliver siRNAs and control infection in vitro, indicating a novel anti-DENV strategy.
36 37 38
Keywords: dengue virus, siRNA delivery, gold nanoparticle
AuNPs deliver anti-Dengue siRNA
39
Introduction
40
Dengue virus (DENV) is a single-stranded, positive-sense RNA virus belonging to the
41
family Flaviviridae. DENV is transmitted to humans primarily by the mosquito vectors, Aedes
42
aegypti and Aedes albopictus (Xi et al., 2008). After DENV binds to host cellular receptors, it is
43
endocytosed and uncoated within the endosome to release its RNA genome into the cytoplasm,
44
where replication of the viral genome and assembly of virions occur (van der Schaar et al.,
45
2008). The common symptoms of DENV infection in humans are characterized by fever,
46
headache, joint and muscle pain and measle-like rashes (Halstead, 2007; Wilder-Smith et al.,
47
2010). Although asymptomatic in most cases, DENV infection can lead to serious disease, such
48
as hemorrhagic fever and systemic shock syndrome that can potentially be fatal. There are four
49
serotypes of DENV (DENV-1, DENV-2, DENV-3 and DENV-4) and illness can be produced by
50
any serotype (Tomashek & Margolis, 2011). Although recovery from infection to one serotype
51
provides lifelong immunity against that particular serotype, subsequent infection by other co-
52
circulating serotypes may cause an antibody-dependent enhancement reaction, which increases
53
the risk of developing severe dengue hemorrhagic fever, shock and even death (Halstead, 1982;
54
Kliks et al., 1989). DENV infection has been reported in more than 100 countries, including the
55
United States (Gubler, 2002; Ramos et al., 2008; Tomashek & Margolis, 2011). A recent report
56
has estimated that 390 million people are infected with DENV worldwide, with 96 million
57
people manifesting clinical symptoms of disease (Bhatt et al., 2013).
58
Since antibody-dependent enhancement hinders the development of an antibody-based
59
vaccine, the development of therapeutics that target replication or expression of the viral genome
60
may serve as promising alternatives to control DENV infection. Anti-host receptor (Alhoot et al.,
61
2011) and anti-DENV (Stein et al., 2011; Subramanya et al., 2010) small interfering RNAs
3
AuNPs deliver anti-Dengue siRNA
62
(siRNAs) have considerably advanced both clinical- and laboratory-based applications as
63
potential DENV therapeutic alternatives (Idrees et al., 2013). However, efficient delivery of
64
siRNAs remains a great challenge for clinical applications. The most common siRNA delivery
65
approach is with a lipid-based transfection reagent, whereby the negatively charged siRNA
66
duplex binds to the cationic liposome transfection reagent, which allows for cell membrane
67
endocytosis, micelle formation and intracellular release of the siRNA into the cytosol (Zuhorn et
68
al., 2002). Although this method is successful for siRNA delivery in vitro, it is not an ideal
69
candidate to use in vivo, as these chemically-based transfection reagents are physiologically toxic
70
and could induce cellular stress or aberrant transcriptional profiles (Torchilin, 2005; Zhong et al.,
71
2008).
72
Recently, gold nanoparticles (AuNPs) have been suggested as promising vehicles to
73
deliver siRNA both in vitro and in vivo (Conde et al., 2012; Conde et al., 2013; Mitra et al.,
74
2013; Yang et al., 2013). Several advantages make them well suited for safe and efficient
75
protection and transport of siRNA: a) biocompatibility (Chithrani & Chan, 2007; Lewinski et
76
al., 2008); b) ease of preparation and facile surface modification (Elbakry et al., 2009; Lee et al.,
77
2011; Song et al., 2010); and c) high siRNA loading capacity. In this study, we constructed novel
78
anti-DENV AuNP-siRNA nanoplexes and found that AuNPs are able to promote siRNA stability
79
and efficiently deliver anti-DENV siRNAs in vitro.
80
4
AuNPs deliver anti-Dengue siRNA
81
Results
82
Antiviral siRNAs efficiently inhibit DENV replication
83
Two antiviral siRNAs (si-1 and si-3) were designed to target the most prevalent dengue
84
serotype, DENV-2. Vero cells were transfected with siRNA (20 nM) using a lipofectamine-based
85
transfection reagent (TR) and subsequently infected with DENV-2 (multiplicity of infection,
86
MOI = 0.1). Sequence scrambled siRNA (siSCRM) was used as a negative control and a
87
previously described anti-DENV siRNA (DC-3) was used as a positive control (Stein et al.,
88
2011). At 48 hr post-infection, the cells were harvested for total RNA extraction, cDNA
89
synthesis and subjected to real-time quantitative polymerase chain reaction (Q-PCR) for the
90
measurement of DENV capsid gene and cellular β-actin gene expression. The Q-PCR results
91
showed that si-1, si-3 and DC-3 significantly reduced DENV-2 capsid gene expression compared
92
to the control, in which cells were treated with TR only (p < 0.001, Fig. 1a). In contrast, siSCRM
93
did not show any inhibitory effect (p = ns, Fig. 1a), suggesting that siRNA inhibition is
94
sequence-specific. To test if infectious viral particles released from the infected cells were
95
reduced with siRNA transfection, the media were quantified by a plaque forming assay (plaque
96
forming unit, PFU) and an immunostaining assay (focus forming unit, FFU), with the latter
97
detecting viral envelope (E) protein expression. Since the DENV-2 genome shares some
98
homology with other DENV-serotypes, the antiviral effect of si-1 and si-3 (20 nM) was also
99
evaluated against the replication of DENV-1, DENV-3 and DENV-4 (MOI = 0.1). Q-PCR
100
analysis showed that si-1 and si-3 were able to inhibit DENV-3 and DENV-4 replication, but
101
showed no inhibitory activity for DENV-1, compared to the control in which cells were treated
102
with TR, without siRNA (p < 0.001 and p = ns, Fig. 1d-f). RNA sequence alignment analysis
103
suggested that the lack of inhibition of si-1 and si-3 against DENV-1 replication might be due to
5
AuNPs deliver anti-Dengue siRNA
104
lower homology between their targeting sequences (approximately 50% and 70% respectively,
105
data not shown). In summary, these results demonstrated that both si-1 and si-3 were able to
106
significantly inhibit replication of DENV serotypes 2, 3 and 4.
107 108
Design and optimization of AuNP complexes for anti-DENV siRNA delivery in vitro
109
Recently, AuNPs have been designed and optimized as delivery vehicles for siRNA both
110
in vitro and in vivo (Conde et al., 2013; Gilleron et al., 2013; Mitra et al., 2013; Yang et al.,
111
2013; Zheng et al., 2012). Optimization of AuNPs has been focused on certain properties that
112
enhance siRNA delivery and reduce possible cytotoxicity. Such properties include surface
113
charge, surface complexity and size of the AuNPs (De Jong & Borm, 2008). In this study, three
114
AuNP-siRNA complexes (anionic, neutral and cationic) were prepared by a novel layer-by-layer
115
(LbL) approach to deliver anti-DENV siRNA into Vero cells. To confirm each layer was
116
successfully coated on the AuNPs, the AuNP complexes were characterized by UV-Vis spectra,
117
DLS and Zeta potential. AuNPs and AuNP-PEI-siRNAs (PEI: polyethylenimine) were measured
118
by UV-Vis, which disclosed a shift in the Surface Plasmon Resonance (SPR) peaks with the
119
addition of siRNA (Fig. 2a). DLS measured the hydrodynamic diameters of the different
120
complexes: AuNPs (12.92 nm), AuNP-PEI (24.97 nm), AuNP-PEI-siRNA (41.79 nm), and
121
AuNP-PEI-siRNA-PEI (43.25 nm), which indicated that the diameters increased with the
122
addition of each layer (Fig. 2b). AuNPs were first coated with an inner layer of HS-PEI (thiol-
123
labeled PEI) via Au-S bonds, rendering the particles cationic. The negatively charged siRNAs
124
were then coated on the AuNP-PEIs through electrostatic interactions, rendering the complexes
125
anionic. Adjusting the concentration of the outer layer PEI with the AuNP-PEI-siRNA
126
complexes (Fig. 2c) altered the overall charge from negative to positive. Zeta potential
6
AuNPs deliver anti-Dengue siRNA
127
determined the surface charges of the complexes: AuNPs (-34.9 mV), AuNP-PEI (46.9 mV),
128
AuNP-PEI-siRNA (-38.8 mV) and AuNP-PEI-siRNA-PEI (53.3 mV) (Fig. 2d, neutral charge
129
data not shown). These results demonstrated the optimization of AuNPs and successful synthesis
130
of AuNP-siRNA complexes.
131
To test the effect of surface charge on siRNA delivery efficiency, we treated Vero cells
132
with anionic (-40 mV), neutral (0 mV) and cationic (+40 mV) AuNP-siRNA complexes (20 nM)
133
for 48 hrs and subsequently infected them with DENV-2 (MOI = 0.1) for an additional 48 hrs. Q-
134
PCR analysis showed that cationic AuNP-si-1 and AuNP-si-3 complexes significantly reduced
135
DENV-2 replication (p < 0.005, Fig. 2e). While anionic AuNP-si-3, but not AuNP-si-1
136
complexes, also inhibited DENV-2 replication compared to the control, in which cells were
137
infected with DENV-2 only (p < 0.05, Fig. 2e). In contrast, neither the neutrally charged AuNP-
138
si-1 nor the AuNP-si-3 complex inhibited viral replication, suggesting inefficient delivery of
139
siRNAs by neutral AuNP-siRNA complexes (Fig. 2e). In summary, these results showed that
140
cationic AuNP-siRNA complexes could effectively deliver anti-DENV siRNAs into cells.
141
To confirm cationic AuNP-siRNAs are able to enter cells, Vero cells were cultured with
142
cationic AuNP-si-1 (80nM) for 48 hrs and subsequently infected with DENV-2 (MOI = 0.1) for
143
an additional 72 hrs. Ultra-thin sections embedded in epoxy resin were imaged using a
144
transmission electron microscope (TEM). The TEM images showed that AuNPs localized within
145
the cytoplasm of Vero cells (Fig. 2f, g and inserts), which implied AuNP complexes were able to
146
deliver siRNAs into the cells.
147 148
Cationic AuNP-siRNA complexes are non-toxic to Vero cells
7
AuNPs deliver anti-Dengue siRNA
149
AuNPs have been noted as biocompatible vehicles for drug delivery (Connor et al., 2005;
150
Shukla et al., 2005). However, they also have been suggested to have some cytotoxicity due to
151
their cationic polarity and surface modifications (Gerber et al., 2013; Goodman et al., 2004). To
152
exclude the possibility that inhibition of DENV-2 replication was due to the AuNP-siRNA
153
complexes causing Vero cell death, we assessed the cytotoxicity of the AuNP-siRNA complexes
154
when exposed to Vero cells. We incubated AuNP-siRNA complexes (+40 mV) with Vero cells
155
at the same experimental conditions and concentrations that were used for the pre-treatment
156
transfection assays, without DENV-2 infection. Cellular morphology was imaged and cell
157
number was quantified by using DAPI-nuclear staining and examined under a confocal
158
microscope. No morphological changes (Fig. 3a, i and ii) or cell number reduction (Fig. 3b) were
159
observed with any AuNP-siRNA treated samples compared to the untreated control. To further
160
confirm this, cellular lactate dehydrogenase (LDH) released from broken cell membrane was
161
also measured. The LDH cytotoxicity assay results showed no significant toxicity (≤ 6.5%
162
cytotoxicity), which was similar with the untreated cell control (2% cytotoxicity, Fig. 3c). These
163
data suggested that Vero cells exposed to AuNP-siRNA complexes have negligible cytotoxicity,
164
which indicated that inhibition of DENV-2 replication was due to siRNA mediated RNA
165
interference.
166 167
Cationic AuNP-siRNA complexes inhibit DENV-2 infection in both pre- and post-infection
168
conditions
169
To further confirm that cationic AuNP-siRNA complexes inhibit DENV-2 replication, we
170
pre-treated Vero cells with cationic AuNP-siRNAs (+40 mV) at 20 nM and 80 nM for 48 hrs and
171
subsequently infected them with DENV-2 (MOI = 0.1) for an additional 48 and 72 hrs, followed
8
AuNPs deliver anti-Dengue siRNA
172
by Q-PCR analysis. The results showed that compared to DENV-2 only infected controls,
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AuNP-si-1 (20 nM) reduced DENV replication approximately 2-fold at both 48 and 72 hrs (p
