Subscriber access provided by : Bibliothèque | Polytechnique Montréal
Article
Nitric Oxide Delivery by Core/shell Superparamagnetic
Nanoparticle Vehicles with Enhanced Biocompatibility
Xuefeng Zhang, Sania Mansouri, D. A. Mbeh, L'Hocine Yahia, Edward Sacher, and Teodor Veres
Langmuir, Just Accepted Manuscript • Publication Date (Web): 15 Aug 2012
Downloaded from http://pubs.acs.org on August 15, 2012
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.
Page 1 of 23
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Langmuir
Nitric Oxide Delivery by Core/shell Superparamagnetic
Nanoparticle Vehicles with Enhanced Biocompatibility
X. F. Zhang,†,
‡, #
S. Mansouri,
†, ‡, #
D. A. Mbeh,
§
L’H. Yahia,
§
E.
Sacher, § and T. Veres †, ‡, *
†
National Research Council of Canada ,75 Boul. de Mortagne, Boucherville, Québec, Canada,
J4B 6Y4
‡
INRS Énergie, Matériaux et Télécommunications, 1650 boulevard Lionel Boulet,Varennes,
Québec, Canada, J3X 1S2
§
École Polytechnique de Montréal, Case Postale 6079, succursale Centre-Ville, Montréal
Québec, Canada, H3C 3A7
*Corresponding author, E-mail: Teodor.Veres@cnrc-nrc.gc.ca; Fax: + 450 641-5105
#
Equal contribution
We report the synthesis of Fe3O4/silica core/shell nanoparticles and their functionalization with
S-nitrosothiols. These nanoparticles are of immense interest due to their nitric oxide (NO) release
capabilities in human alveolar epithelial cells. Moreover, they act as large storage reservoirs of
NO that can be targeted magnetically to the specific site with a sustainable release of NO up to
50 h. Such nanoparticles provide an enhancement of the biocompatibility with the released NO,
while allowing intracellular accumulation ascribed to their small size.
1
ACS Paragon Plus Environment
Langmuir
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Page 2 of 23
1 Introduction
Nitric oxide (NO) is an extremely important biological agent that has the abilities to protect the
cardiovascular tissue by inhibiting thrombosis and enzymes, to mediate neurotransmission, and
to repair wounds.(1-3) Development of nitric oxide (NO) delivery vehicles has been attracting
considerable attention due to such physiological features. (4-13) However the delivery of NO
molecules at specific tissue sites that provide therapeutically effective release while reducing
toxic side-effect of delivery vehicles still remains challenging. The ideal delivery vehicle should
be an integration of biocompatibility, targeted release, and the ability to pass through the cell
membrane wall for efficient cell uptake. In this communication, Fe3O4/silica core/shell
nanoparticles have been synthesized to chemically store and release NO by functionalizing the
silica shells with S-nitrosothiols that can spontaneously release NO through the homolytic bond
cleavage
of
the
S-N
bond
under
physiological
conditions.(14-17)
The
integrated
superparamagnetic cores of such nanoparticles exhibit superior potential for the magnetic
targeting, while the silica shells increase their biocompatibility and allow a better permeability
for cellular uptake.
2 Experimental details:
2.1 Materials
Oleic acid (OA, 90%), 1-hexaneol anhydrous (99%), octyl ether (98%), ammonia solution
(NH4OH, 28-30 wt % in water), Triton X-100, hexane (95%), cylcohexane (99.5%), and
tetraethoxysilane (TEOS, 99.999%), Ru(bpy)32+ complex dyes were purchased from SigmaAldrich Inc. Iron pentacarbonyl (99.5%) was purchased from Strem Chemicals, Inc.
(Newburyport, MA). N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAP3, ≥ 90%), 3mercaptopropyltrimethoxysilane (≥ 97%) was purchased from Gelest (Tullytown, PA).
2
ACS Paragon Plus Environment
Page 3 of 23
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Langmuir
2.2 Synthesis of core/shell Fe3O4/silica(SH) nanoparticles
Fe3O4 nanoparticles, with a thin protective layer of oleic acid (Fe3O4/OA), were synthesized
based on a thermal decomposition process.(18) 20 mL of octyl ether were mixed with 1.92 mL
oleylamine at room temperature for ~10 minutes, and subsequently heated to 100 °C within 20
min under the Ar flow. At 100 °C, 0.4 mL of iron pentacarbonyl were injected, the temperature
was increased to reflux for 2 h and then cooled to room temperature by removing the heating
mantle. The product was precipitated by adding excess anhydrous ethanol, and separated by
centrifugation (9000 rpm), and further washed with cyclohexane and ethanol for at least three
times. The core/shell Fe3O4/silica nanoparticles were synthesized in a water-in-oil
microemulsion. 0.5 ml of 1 mg/mL Fe3O4/OA nanoparticles in cyclohexane were injected into a
mixture of 1.77 g of Triton X-100, 1.6 mL of anhydrous 1-hexanol and 7 mL of cyclohexane
under a strong vortex for about 1 h, and then 0.5 mL of ammonia solution (6 % ammonia
solution) were added, with stirring for another 1 h. To synthesize the fluorescence Fe3O4/silica
nanoparticles, 20 mg Ru(bpy)32+ complex dyes were added in the above mixture with an
additional mixing for 30 min. Twenty five µL of TEOS were added, and left to react for 24 h.
Subsequently, 2 µL of 3-mercaptopropyltrimethoxysilane were added to the reaction mixture,
and left for another 24 h. The resultant thiol-functionalized Fe3O4/silica nanoparticles (was
denoted as Fe3O4/silica(-SH) nanoparticles. The Fe3O4/silica(-SH) nanoparticles were separated
by centrifugation at 9000 rpm, washed with ethanol and distilled water, and dried under vacuum
for use.
2.3 Synthesis of core/shell Fe3O4/silica(-SNO) nanoparticles
The addition of NO groups, transforming –SH groups to –SNO groups of Fe3O4/silica(-SH) NPs,
occurs by reacting the -SH functional groups with t-butyl nitrite. The detailed procedures are: 20
3
ACS Paragon Plus Environment
Langmuir
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
mg Fe3O4/silica(-SH) nanoparticles and excess t-butyl nitrite (about 20 mg) were dispersed in 10
mL 10% methanol/90% toluene (v/v) and stirred for 24 h at room temperature under N2 flow.
The resultant nanoparticles with –SNO groups were centrifuged at 9000 rpm, and washed three
times with anhydrous ethanol/and de-ionized water, then rapidly dried in vacuum for 5 min
(longer drying time will cause –SNO decomposition), and finally stored at low temperature (-20
o
C) under light-shielded condition.
2.4 Characterizations of the nanoparticles
The microstructures of the nanoparticles were characterized by X-ray diffraction (Siemens D500 X-ray diffractometer with CuKα (λ=0.154 nm), and transmission electron microscopy
(Hitachi S-4700 at30 kV and JEOL 2010F at 200 kV). The surface chemistry was characterized
by Fourier transmission infrared (FTIR) spectra, collected with a Nicolet Fourier
spectrophotometer at 600 and 4000 cm-1. Magnetic properties were determined with a Quantum
Design PPMS model 6000 magnetometer.
2.5 Quantitive evaluation of the NO release of Fe3O4/silica(-SNO) nanoparticles
Detection of the released NO molecules from the nanoparticles was carried out by amperometric
analysis, using the Nitric Oxide Detector (World Precision Instruments). The ISO-NOP sensor
(World Precision Instruments Ltd.) was calibrated by the additions of 50 µM (100 µl for each
step) NaNO2 into 20 mL 0.1 M H2SO4 + 0.1 M KI solution. The detection sensitivity was
determined to be 3.478 pA/nM at 37 oC. The temperature-triggering release of the NO molecules
from the decomposition of the S-Nitrosothiol groups in Fe3O4/silica (-SNO) nanoparticles was
done at normal body temperature (37 oC), under light expose. The details are: 0.7 mg
nanoparticles were dispersed into 1 mL PBS 7.2 buffer, forming a stable suspension, and then
rapidly injected into 19 mL PBS 7.2 buffer, at which time the ISO-NOP sensor had reached a
4
ACS Paragon Plus Environment
Page 4 of 23
Page 5 of 23
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Langmuir
low and stable current level. The NO probe was immersed about 2 cm into the suspension, with
magnetic stirring at 600 rpm, and the measurement temperature was fixed at 37 oC.
2.6 Cell culture
The in-situ NO release and the cytotoxicity of the Fe3O4/silica(-SNO) nanoparticles were
evaluated using adenocarcinomic human alveolar basal epithelial cells (A549, American Type
Culture Collection, ATCC, USA). The medium used was Ham’s F-12 (ATCC, USA)
supplemented with penicillin (100 IU/mL), streptomycin (100 µg/mL) and 10% fetal bovine
serum (FBS). The cells were cultured at a density of 1×105 cells per 1 mL of medium in 24-well
culture plates at 37°C in a 5% CO2 atmosphere. After 20 h of culture, the medium in the wells
was replaced with fresh medium containing Fe3O4/silica(-SNO) nanoparticles (1, 5 and 10
µg/mL) and was further cultured for 48 h. In control cultures, the cells were placed in the
medium, without nanoparticles, at the same cell density.
2.7 Intracellular release of NO
Cell line A549 (1×105 cells/mL) were incubated with Fe3O4/silica(-SNO) nanoparticles (10
µg/mL) for 3, 24 and 48 h. After 3 washes with culture medium, 10 µm of 4-amino-5methylamino-2`, 7`-difluorofluorescein, DAF-FM (Sigma), were added and incubated for 2 h at
37°C. After incubation, the cells were then washed three times with culture medium.
Fluorescence images were captured with a computer-controlled charged-coupled device (CCD)
camera using an image-capture system consisting of a Nikon Eclipse microscope equipped with
a Filter set and a ×20 objective,
5
ACS Paragon Plus Environment
Langmuir
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2.8 In-vitro cell viability
The cell viability test was carried out via the reduction of the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) reagent (Invitrogen). After 48 h of culture with the
Fe3O4/silica(-SNO) nanoparticles (1, 5 and 10 µg/mL), 100 µL of MTT dye solution (5 mg/mL in
phosphate buffer pH-7.4) were added to each well and incubated for 4 h at 37°C and 5% CO2.
The medium was removed and formazan crystals were solubilized with 150 µL of dimethyl
sulfoxide (DMSO). Absorbance of each well was read using a spectrophotometer (Biotek, USA)
at 570 nm, and the relative cell viability (%), related to control wells containing cell culture
medium without nanoparticles, was calculated as [A]test/[A]control × 100. Three replicates were
measured, and the results presented as mean ± standard deviation.
3 Results and Discussion
Figure 1 (a) shows the TEM image of Fe3O4/silica(-SH) nanoparticles. The nanoparticles are all
spherical in shape with a narrow size distribution consisting for a core diameter of 10 nm and a
shell thickness of 20 nm. The conjugation of NO molecules is carried out by reacting the -SH
functional groups of Fe3O4/silica(-SH) nanoparticles with t-butyl nitrite, as shown in Figure 1(c),
and the product, Fe3O4/silica (-SNO) nanoparticles, are as shown in the TEM image of Figure 1
(b). The surface functional groups of the silica shells have been confirmed by Fourier transform
infrared (FTIR) spectra, as shown in Figure 2. The formation of the -S-NO groups on the surface
of Fe3O4/silica nanoparticles is demonstrated by the presence of the characteristic peaks of -SN=, -CH2-S-, -N=O at 764, 1237 and 1505 cm-1. (19-22) However, the transformation rate
(loading rate) from –SH and -S-NO groups cannot be quantitatively estimated. Although a small
6
ACS Paragon Plus Environment
Page 6 of 23
Page 7 of 23
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Langmuir
amount of -SH groups are unavoidably remained; the reaction time (24 hours) and excess usage
of t-butyl nitrite assure that loading of -S-NO groups approaches its saturation.
The surface functional groups and the shell topology of silica coatings weakened the magnetic
interactions between the nanoparticles, thus resulted in nanoparticles having a good monodispersibility and stability in aqueous solution. The as-synthesized Fe3O4/silica (-SNO)
nanoparticles exhibit typical superparamagnetic behavior as shown in Figure 3 (a)-(c). The
hysteretic loops present a saturation magnetization (Ms) of ~3.2 emu/g and a coercive field (HC)
of ~19 Oe at 5 K, and ~2.7 emu/g and zero Oe at 300 K. Considering the saturation
magnetization (Ms) of 73 emu/g for Fe3O4/OA nanoparticles, (23) it is estimated that the
nonmagnetic silica compositions is about
96.8 wt%. The ferromagnetic-superparamagnetic
transition onset appears at around 191 K in the zero-field-cooling (ZFC) and field-cooling (FC)
magnetization curves at an applied magnetic field of 100 Oe.
Figure 4 shows a series images of the magnetic manipulation process of the Fe3O4/silica
nanoparticles doped by Ru(bpy)32+ complex dyes flowing in a plastic tube with 0.75 mm in
diameter. The concentration of Fe3O4/silica nanoparticles and its flow velocity were fixed to be
50 µg/ml and 20 cm/s that roughly based on the normal blood circulation in body. (24) When a
magnetic field of ~1 T was applied on one side of the plastic tube, we found the Fe3O4/silica
nanoparticles were rapidly captured. Remove the magnetic field, the Fe3O4/silica nanoparticles
were immediately released without any physical adsorption on the tube wall. These advantages
make the Fe3O4/silica nanoparticles own potentials for the magnetically targeted delivery of NO
molecules at specific sites.
7
ACS Paragon Plus Environment
Langmuir
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Quantitative detection of NO release of the nanoparticles was carried out by amperometric
analysis using the Nitric Oxide Detector (ISO-NOP sensor, World Precision Instruments Ltd.).
The results revealed an immediate NO release following the addition of nanoparticles. From
Figure 5 (a), it is evident that the release rate of NO is relatively stable, exhibiting an initial peak
of 46.7 nM/mg at 6.55 h, followed by a gradual decrease with time, for up to 50 h. The
cumulative NO release amount is approximately 1.34 µmol NO per 1 mg nanoparticles with a
standard deviation of 5.7 % for 3 sample batches. It should be noted that, at 50 h, the cumulative
release is already close to a plateau, accompanying a very low release rate, which probably
implies a total NO storage amount in nanoparticles. The released capacity of Fe3O4/silica(-SNO)
nanoparticles is comparable to that of silica(-SNO) nanoparticles (0.09-4.39 µmol/mg). (10) The
duration of NO release is about 50 h, with a half-life (t1/2) of 16.9 h. It is noteworthy that the
samples stored for up to 4 months, exhibited release kinetics (time and cumulative total amount)
comparable to that of the fresh samples. Additionally, we addressed the stability of the product at
room temperature, as shown in Figure 5 (b), indicating that only 18.3% of cumulative NO was
released at 20 oC after 50 h compared with that at 37 oC.
One critical aspect of NO delivery vehicles is their permeability through cell membranes, which
can significantly enhance the cellular uptake at targeted sites. Human alveolar epithelial cells
were used to assess this issue. Intracellular fluorescence imaging of NO production was achieved
using a commercial NO indicator, 4-amino-5-methylamino-2`, 7`-difluorofluorescein (DAF-FM)
with a detection limitation of ~3 nM.(25) Figure 6 presents the bright-field and fluorescence
microscopy images of cells incubated by 10 µg/mL Fe3O4/silica(-SNO) nanoparticles with
various time.
8
ACS Paragon Plus Environment
Page 8 of 23
Page 9 of 23
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Langmuir
After 3 h of incubation, the fluorescence inside the cells was weak as an indication of low NO
uptake. The nanoparticles internalized into the cells were quite limited, and the released NO was
therefore distributed mainly in the extracellular medium that has been washed away. In
comparison, after 24 h of incubation, the Fe3O4/silica(-SNO) nanoparticles were localized in the
whole cell (cytoplasm, subcellular vesicles and even nucleus), as indicated by the visible
fluorescence micrograph [Figure 6 (d)]. The uptake process of nanoparticles by the cells occurs
through endocytosis that requires a nanoparticle size smaller than 100 nm. The intracellular
release of NO was attributed to the distinct characteristics of the nanoparticles, such as the monodispersibility and the small size distribution of around 40 nm, which can enhance the cellular
uptake and accumulation. However, the cells did not present obvious fluorescence intensity after
48 h incubation in Figure 6(f), indicating that the Fe3O4/silica(-SNO) nanoparticles cannot
sustain a detectable release of NO in cells over 48 h, which is consistent with that measured by
amperometric analysis, in Figure 5.
The viability of human alveolar epithelial cells, affected by the NO released from the
Fe3O4/silica(-SNO) nanoparticles, was quantitatively determined by the MTT assay, as shown in
Figure 7. Although many studies have reported that NO has no influence on cell viability or
manifold cytotoxicity,(26-28) our experiments indicate that at low concentrations of
nanoparticles (1 and 5 µg/mL), the viability was slightly increased compared with the control cell
lines, and gradually decreased as the concentrations increase, presenting a viability of 70 % at
100 µg/mL. It has been demonstrated that the cytoprotective role for low NO concentration is to
act directly as an antioxidant to scavenge peroxyl radicals. Moreover, NO can significantly
inhibit lipid peroxidation by ferrous compounds/H2O2 and reactive oxygen intermediates like
Fe2+.(29-33) From Figure 7, the cell viability, as a percentage of Fe3O4/silica(-SNO)
9
ACS Paragon Plus Environment
Langmuir
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Page 10 of 23
nanoparticles, was higher than the Fe3O4/silica(-SH) nanoparticles, suggesting that released NO
from S-nitrosothiols can suppress the negative effects of silica-coated nanoparticles.
Additionally, the Fe3O4/silica(-SNO) have shown a better cell viability compared with that of
Fe3O4/silica nanoparticles with primary amine groups in our recent study. (23) These results
suggest that S-nitrosothiols, as surface functional groups of biomedical vehicles, can improve
biocompatibility.
In summary, we reported the synthesis of Fe3O4/silica (-SNO) core/shell nanoparticles and the
concept of their use as NO delivery vehicles. Their relatively small size provides the possibility
to permeate through the cell membrane to subsequently release the molecule of interest. The
surface modification of the nanoparticles with S-nitrosothiols increased their biocompatibility.
In addition, such hybrid nanostructures, with superparamagnetic cores, have great application
potential in magnetically targeted manipulation, magnetic resonance imaging and hyperthermia.
10
ACS Paragon Plus Environment
Page 11 of 23
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Langmuir
Acknowledgment. The work was jointly supported by Natural Sciences and Engineering research
Council of Canada, the Canadian Institutes of Health Research and the National Research Council of
Canada, Industrial Materials Institute (IMI-NRC). We are grateful to Nitric Medical Devices (NMD) Inc.
for financial support, and Drs. Blaise Gilbert and Dr. Omar Quraishi for the insightful advice on the use
of magnetic carriers for biomedical applications. We are grateful to Dr. Lidija Malic for the technical
revision and significant discussion.
11
ACS Paragon Plus Environment
Langmuir
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Page 12 of 23
References
(1)
Flavahan, N. A. Atherosclerosis or lipoprotein-induced endothelial dysfunction. Potential
mechanisms underlying reduction in EDRF/nitric oxide activity. Circulation 1992, 85,
1927-1938.
(2)
Vane, J. R.; Anggard, E. E.; Botting, R. M. Regulatory functions of the vascular
endothelium. N. Engl. J. Med. 1990, 323, 27-36.
(3)
Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Nitric Oxide: Physiology, Pathophysiology
and Pharmacology. Pharmacol. Rev. 1991, 43, 109-142.
(4)
Keefer, L. K. Biomaterials: Thwarting Thrombus. Nat. Mater. 2003, 2, 357-358.
(5)
Zhang, H.; Annich, G.M.; Miskulin, J.; Stankiewicz, K.; Osterholzer, K.; Merz, S. I.;
Bartlett, R. H.; Meyerhoff, M. E. Nitric Oxide-Releasing Fumed Silica Particles: Synthesis, Characterization, and Biomedical Application. J. Am. Chem. Soc. 2003, 125,
5015-5024.
(6)
Ghaffari, A.; Neil, D. H.; Ardakani, A.; Road, J.; Ghahary, A.; Miller, C. C. A Direct
Nitric Oxide Gas Delivery System for Bacterial and Mammalian Cell Cultures, Nitric
Oxide 2005, 12, 129-140.
(7)
Polizzi, M. A.; Stasko, N. A.; Schoenfisch, M. H. Water-Soluble Nitric Oxide-Releasing
Gold Nanoparticles, Langmuir, 2007, 23, 4938-4943
(8)
Robbins, M. E.; Hopper, E. D.; Schoenfisch, M. H. Synthesis and Characterization of
Nitric Oxide-Releasing Sol-Gel Microarrays, Langmuir, 2004, 20, 10296-10302.
(9)
Shin, J. H.; Metzger, S. K.; Schoenfisch, M. H. Synthesis of Nitric Oxide-Releasing Silica
Nanoparticles. J. Am. Chem. Soc. 2007, 129, 4612-4619.
12
ACS Paragon Plus Environment
Page 13 of 23
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Langmuir
(10) Riccio, D. A.; Nugent, J. L.; Schoenfisch, M. H.; Stöber Synthesis of Nitric OxideReleasing S-Nitrosothiol-Modified Silica Particles. Chem. Mater. 2011, 23, 1727–1735.
(11) Jo, Y. S.; van der Vlies, A. J.; Gantz, J.; Thacher, T. N.; Antonijevic, S.; Cavadini, S.;
Demurtas, D.; Stergiopulos, N.; Hubbell, J. A. Micelles for delivery of nitric oxide. J. Am.
Chem. Soc. 2009, 131, 14413-14418.
(12) Pinto, M. L.; Rocha, J.; Gomes, J. R. B.; Pires, J. Slow Release of NO by Microporous
Titanosilicate ETS-4. J. Am. Chem. Soc. 2011, 133, 6396-6402.
(13) Riccio, D. A.; Dobmeier, K. P.; Hetrick, E. M.; Privett, B. J.; Paul, H. S.; Schoenfisch, M.
H. Biomaterials 2009, 30, 4494-4502.
(14) Al-Sa’doni, H.; Ferro, A. S-Nitrosothiols: a Class of Nitric Oxide-Donor Drugs. Clinical
Science 2000, 98, 507-520.
(15) Frost, M. C.; Meyerhoff, M. E.; Synthesis, Characterization, and Controlled Nitric Oxide
Release from S-nitrosothiol-derivatized Fumed Silica Polymer Filler Particles. J. Biomed.
Mater. Res. A. 2005, 15, 409-419.
(16) Josephy, P. D.; Rehorek, D.; Janzen, E. D. Electron Spin Resonance Spin Trapping of
Thiyl, Radicals from the Decomposition of Thionitrites. Tetrahedron Lett. 1984, 25, 16851688.
(17) Oliveira, M. G.; Shishido, S. M.; Seabra, A. B.; Morgon, N. H. Thermal Stability of
Primary S -Nitrosothiols: Roles of Autocatalysis and Structural Effects on the Rate of
Nitric Oxide Release. J. Phys. Chem. A 2002, 106, 8963-8970.
(18) Woo, K.; Hong, J.; Choi, S.; Lee, H. W.; Ahn, J. P.; Kim, C. S.; Lee, S. W. Easy Synthesis
and Magnetic Properties of Iron Oxide Nanoparticles. Chem. Mater. 2004, 16, 2814-2818.
13
ACS Paragon Plus Environment
Langmuir
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Page 14 of 23
(19) Goto, K.; Hino, Y.; Kawashima, T.; Kaminaga, M.; Yano, E.; Yamamoto, G.; Takagi, N.;
Nagase, S. Synthesis and crystal structure of a stable S-nitrosothiol bearing a novel steric
protection group and of the corresponding S-nitrothiol. Tetrahedron Letters 2000, 41,
8479-8483.
(20) Williams, D. L. H. The Chemistry of S-Nitrosothiols. Acc. Chem. Res. 1999, 32, 869-876.
(21) The Sadtler Handbook of Infrared Spectra, p82, edited by Dr. John R. Ferraro.
(22) Perissinotti, L. L.; Leitus, G.; Shimon, L.; Estrin, D.; Doctorovich, F. A Unique Family of
Stable and Water-Soluble S-Nitrosothiol Complexes. Inorganic Chem. 2008, 47, 47234733.
(23) Zhang, X. F.; Mansouri, S.; Clime, L.; Ly, H. Q.; Yahia L. ’H.; Veres, T. Fe3O4-silica
core–shell nanoporous particles for high-capacity pH-triggered drug delivery. J. Mater.
Chem. 2012, 22, 14450-14457.
(24) Wexler, L.; Bergel, D. H.; Gabe, I. T.; Makin, G. S.; Mills, C. J. Velocity of Blood Flow in
Normal Human Venae Cavae. Circulation Research 1968, 23, 349-359.
(25) Kojima, H.; Urano, Y.; Kikuchi, K.; Higuchi, T.; Hirata, Y.; Nagano, T. Fluorescent
Indicators for Imaging Nitric Oxide Production. Angew. Chem. Int. Ed. 1999, 38, 32093212.
(26) Sun, M. H.; Han, X. C.; Jia, M. K.; Jiang, W. D.; Wang, M.; Zhang, H.; Han, G.; Jiang, Y.
Expressions of Inducible Nitric Oxide Synthase and Matrix Metalloproteinase-9 and Their
Effects on Angiogenesis and Progression of Hepatocellular Carcinoma. World J
Gastroenterol 2005, 11, 5931-5937.
(27) Lipton, S. A.; Choi, Y. B.; Pan, Z. H.; Lei, S. Z.; Chen, H. S. V.; Sucher, N. J.; Loscalzo,
J.; Singel, D. J.; Stamler, J. S. A Redox-based mechanism for the neuroprotective and
14
ACS Paragon Plus Environment
Page 15 of 23
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Langmuir
neurodestructive effects of nitric oxide and related nitroso-compounds. Nature 1993, 364,
626-632.
(28) Bonfoco, E.; Krainc, D.; Ankarcrona, M.; Nicotera, P.; Lipton, S. A. Apoptosis and
Necrosis: Two Distinct Events Induced, Respectively, by Mild and Intense Insults with Nmethyl-D-aspartate or Nitric Oxide/Superoxide in Cortical Cell Culture.
Proc. Natl.
Acad. Sci. USA 1995, 92, 7162-7166.
(29) Wink, D. A.; Hanbauer, I.; Grisham, M. B.; Laval, F., Nims, R. W.; Cook, J.; Pacelli, R.;
Liebmann, J.; Krishna, M.; Ford, P. C.; Mitchell, J. B. The chemical biology of NO:
Insights into regulation, protective and toxic mechanisms of nitric oxide. Curr. Top. Cell.
Regul. 1996, 34, 159-187.
(30) Kanner, J.; Harel, S.; Granit, R.; Arch. Nitric Oxide as an Antioxidant. Biochem. Biophys.
1991, 289, 130-136.
(31) Rubbo, H.; Radi, R.; Trujillo, M.; Telleri, R.; Kalyanaraman, B.; Barnes, S.; Kirk, M.;
Freeman, B. A. Nitric Oxide Regulation of Superoxide and Peroxynitrite-dependent Lipid
peroxidation. Formation of Novel Nitrogen-containing Oxidized Lipid Derivatives. J. Biol.
Chem. 1994, 269, 26066-26075.
(32) Rauhala, P.; Sziraki, I.; Chiueh, C. C. Peroxidation of brain lipids in vitro: Nitric oxide
versus hydroxyl radicals. Free Radical Biol. Med. 1996, 21, 391-394.
(33) Hayashi, K.; Noguchi, N.; Niki, E. Action of Nitric Oxide as An Antioxidant Against
Oxidation of Soybean Phosphatidylcholine Liposomal Membranes. FEBS Lett. 1995, 370,
37-40.
15
ACS Paragon Plus Environment
Langmuir
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Page 16 of 23
Figure 1 (a) and (b) TEM image of Fe3O4/silica(-SH) nanoparticles and Fe3O4/silica(-SNO)
nanoparticles; (c) scheme of nitric oxide grafting, showing protocol that transform -SH
functional groups to -SNO groups of Fe3O4/silica(-SH) nanoparticles by reacting the -SH
functional groups with t-butyl nitrite.
16
ACS Paragon Plus Environment
Page 17 of 23
1043
~764
-S-N=
1108
-OH
-S-N=O
1237 1505
1636
CH2-S
%/Transmittance
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Langmuir
(c)
(b)
(a)
800
1200
1600
-1
Wavenumbers (cm )
2000
Figure 2 Fourier transmission infrared (FTIR) spectra of (a) Fe3O4/silica(-OH), (b) Fe3O4/silica(-SH) and
(c) Fe3O4/silica(-SNO) nanoparticles.
17
ACS Paragon Plus Environment
Langmuir
4
1.5
10 K
Magnetication (emu/g)
Magnetication (emu/g)
300 K
2
0
-2
-4
-50000 -25000
0
25000
(a)
50000
10 K
1.0
300 K
0.5
0.0
-0.5
-1.0
-1.5
-100
Magnetic field (Oe)
Magnetization (a.u.)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Page 18 of 23
-50
0
50
Magnetic field (Oe)
(b)
100
191 K
(c)
0
50
100 150 200 250 300
Temperature (K)
Figure 3 Magnetic properties of Fe3O4/silica(-SNO) nanoparticles. (a)-(b) Hysteresis loops and
the enlargements at origin at 5 and 300 K; (c) ZFC-FC magnetization curves cooled under an
applied field of 100 Oe at 10 K.
18
ACS Paragon Plus Environment
Page 19 of 23
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Langmuir
Figure 4 Magnetic manipulation of 100 µl/ml Fe3O4/silica nanoparticles doped by Ru(bpy)32+
complex dyes flowing in a plastic tube with 0.75 mm in diameter. (a) Optical image, (b)
fluorescence image under green optical irradiation, (c) Add a small magnet beside the tube,
showing an immediate capture of nanoparticles (green color), (d) and (e) As the capture time
prolonging, more and more nanoparticles were captured, (f) Remove the magnet, showing a
rapid release of nanoparticles.
19
ACS Paragon Plus Environment
Langmuir
50
1.5
Nitric oxide release (nmol/mg)
1.34 µmol/mg
40
1.2
30
0.9
20
0.6
t1/2=16.9 hrs
10
0.3
0
Cumulative release (µmol/mg)
0.0
0
10
20
30
40
50
Time (hrs)
Nitric oxide release (nmol/mg)
10
0.3
8
0.2
6
4
0.1
2
0
Cumulative release (µmol/mg)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Page 20 of 23
0.0
0
10
20
30
40
50
Time (hrs)
Figure 5 quantitative evaluation of NO release of Fe3O4/silica(-SNO) nanoparticles in PBS 7.4
buffer (a) at 37 oC and (b) 20 oC.
20
ACS Paragon Plus Environment
Page 21 of 23
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Langmuir
Figure 6 Bright-field and fluorescence microscopy images of NO release detection of 10 µg/mL
Fe3O4/silica(-SNO) nanoparticles in cells with various incubation of (a)-(b) 3 h; (c)-(d) 24 h and
(e)-(f) 48 h. Bright-field and fluorescence microscopy images of NO detection of cells without
nanoparticles after incubation of (g)-(h) 3 h; (i)-(j) 24 h and (k)-(l) 48 h.
21
ACS Paragon Plus Environment
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cell Viability Percentage (%)
Langmuir
120
Page 22 of 23
Fe3O4/silica(-SNO)
Fe3O4/silica(-SH)
100
80
60
40
20
0
1
50
5
10
Concentration (µg/mL)
100
Figure 7 Cell viability percentage incubated after 48 h by Fe3O4/silica(-SNO) nanoparticles with
various concentrations of 0, 1, 5, 10, 50, and 100 µg/mL. Control cell viability is normalized to
be 100%.
22
ACS Paragon Plus Environment
Page 23 of 23
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Langmuir
Table of Contents Graphic
23
ACS Paragon Plus Environment