DOI 10.1007/s10517-015-2794-z
500
Bulletin of Experimental Biology and Medicine, Vol. 158, No. 4, February, 2015
NANOTECHNOLOGIES In Vitro Effects of Nanosized Diamond Particles on Macrophages V. A. Shkurupy*,**, S. A. Arkhipov*,**, D. V. Neshchadim*, E. S. Akhramenko*, and A. V. Troitskii* Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 158, No. 10, pp. 503-506, October, 2014 Original article submitted January 18, 2014 The effects of synthetic diamond nanoparticles (4-6 nm) on mouse macrophage biotropism and biocompatibility and the modulation of the macrophage functions (expression of IL-1α, TNF-α, GM-CSF, bFGF, and TGF-β) by nanoparticles in different concentrations were studied in vitro during exposure of different duration. Macrophage endocytosis of nanodiamonds increased with increasing the concentration of nanoparticles in culture and incubation time. Nanodiamonds exhibited high biotropism and biocompatibility towards macrophages; in doses of 10-20 μg/ml, they induced expression of GM-CSF and TGF-β, inhibited expression of bFGF, and did not stimulate IL-1α and TNF-α. These data indicate that nanodiamond capture by macrophages in the studied experimental model led to modulation of the functional status of macrophages that determine their capacity to stimulate reparative processes without increasing proinflammatory and profibrogenic status. Key Words: nanodiamonds; macrophages; biocompatibility; cytokine expression; in vitro
Biocompatibility is one of the most important characteristics of nanosized artificial materials for medicine [3]. Artificial nanosized diamonds (ND) is a prospective nanomaterial [1,6,8,9]. However, the data on ND biotropism are insufficient for their wide use in pharmacy and medical practice because of their probable effects which can become risk factors in certain diseases [2,5,7,8]. Despite obvious chemical inertness, ND are not biologically inert. Being captured by macrophages, they inhibit expression of chemokine CCL2 and PDGF genes in these cells [10]. Macrophages capture many corpuscular objects and are involved in all pathological processes in mammals. We studied the effects of ND particles on the viability and some basic functions of macrophages in vitro. *Research Center of Clinical and Experimental Medicine, Siberian Division of the Russian Academy of Medical Sciences, Novosibirsk; **Novosibirsk State Medical University, Russia. Address for correspondence: [email protected]. S. A. Arkhipov
MATERIALS AND METHODS Two-month-old male BALB/c mice (21-22 g) from Breeding Center of Institute of Cytology and Genetics, Russian Academy of Science were used in the study. Peritoneal macrophages (MP) were isolated after the animals were sacrificed by cervical dislocation under ether narcosis [5]. The cells from peritoneal transsudate were pre-cultured (37oC, 3 h) on slides for MP adhesion (106 cells in 1.5 ml medium 199 with 10% fetal calf serum, FCS). Artificial ND (4-6 nm; UDA-V-GO brand; Research and Production Association “Altai”) were used in the study. Bioactivity of ND towards peritoneal MP was evaluated 1, 3, 24, and 48 h after ND addition to the primary 24-h MP cultures. Sterile suspension (1.6%) of ND in bidistilled water was added into culture medium (medium 199 with 10% FCS) to the final concentrations of 10, 20, 30, and 60 μg/ml. The suspension was then
0007-4888/15/15840500 © 2015 Springer Science+Business Media New York
V. A. Shkurupy, S. A. Arkhipov, et al.
thoroughly mixed and exposed to ultrasonic disintegration (MUZA-0.1/22-M device) for 10 sec at 75 W. The viability of MP (percentage of living cells) in cultures was evaluated by trypan blue staining [5]. Activation of MP was evaluated by activation index corresponding to the area (square pixels) of MP flattened after ND capturing. ROS production was indirectly evaluated by the NBT test [5]. The content of formazan formed in MP was evaluated in arbitrary units (stained area in square pixels) in the microphotographs made under an AxioVision Z1 microscope (Carl Zeiss) at ×400 using VideoTest-Morpho 3.2 software. ROS production by MP was evaluated by ROS production index, equal to the product of the area of MP formazan binary images and the percentage of formazan-containing MP (for respective cultures). Phagocytic activity of MP was evaluated by the phagocytic activity index equal to the product of the phagocytic number and the phagocytic index (percentage of MP with endocytic vacuoles) of the respective culture. ND particles were detected in MP endosomes using combined illumination of the preparations – in transmitting light and induced fluorescence simultaneously [5]. Immunohistochemical studies of MP were carried out by the indirect method. ND (20 μg/ml) were added into 24-h MP cultures according to the same protocol as in studies of the cytophysiological characteristics. Macrophages were cultured with ND for 48 h at 37oC. Macrophage expression of IL-1α, GM-CSF, TNF-α, bFGF, and TGF-β was detected using monoclonal antibodies: anti-mouse IL-1α (Hamster IgG1, λ; Becton Dickinson), anti-mouse GM-CSF (isotype: Rat IgG2a; clone MP1-22E9; Becton Dickinson); anti-mouse TNF-α (Rat IgG1; clone MP6-XT22; Becton Dickinson); anti-TGFbeta (polyclonal antibody, LS-B5663-50; LifeSpan BioSciences); anti-bFGF (polyclonal antibody, bs bs-0217R; Bioss). Rat first antibodies were visualized by anti-rat Ig HRP Detection Kit (Cat. #552013; BD Pharmingen). Rabbit first antibodies were visualized by ImmPRESS anti-rabbit Ig Polymer Detection Kit – Vector Labs MP-7401-15. Hamster first antibodies were visualized by biotinilated anti-hamster antibodies (Cat. #550335; BD Pharmingen) and streptavidin-HRP from Novocastra 250 immunocytochemistry kit. The results were statistically processed by variational statistical methods. The data were presented as the arithmetic means and errors in the means. The significance of differences in the means was evaluated by the nonparametric Mann–Whitney test. The results were statistically processed by Statistica 7.0 software (StatSoft Inc.).
RESULTS Culturing of peritoneal MP in a medium with ND particles in concentrations of 10-30 μg/ml caused no
501 changes in MP viability parameters irrespective of the duration of culturing (Table 1). The level of viable cells decreased by 33% after MP culturing in a medium with 60 μg/ml ND for 48 h. The percentage of MP with endocytic vacuoles containing ND increased with increasing the duration of culturing and ND concentration (Table 1). Endocytic activity index increased with increasing the duration of culturing and ND concentration in the medium; however, after 24 h of culturing it little depended on ND concentration and seemed to be determined by only endocytic capacity of the MP vacuolar system and their capacity to form endocytic vacuoles. Endocytic vacuoles containing ND often fused to form much larger endosomes (Table 1). The capacity of MP to flattening is an integral indicator of their activity associated with the cytoskeleton conformation during realization of the endocytic function and its “energy support” [4-6]. Capture of ND by MP was conjugated with an increase in the counts of flattened cells during the very first hour of their reactions with ND, and the dynamics of this process correlated with ND concentrations in culture medium. However, the involvement of MP in the flattening process was less dynamic after 3 h of culturing and until 48 h of culturing. The percentage of flattened MP decreased by 42% after 48 h of culturing in medium with 60 μg/ml ND, presumably because of a 33% decrease in the level of viable MP during this period (Table 1). Presumably, this was a pool of cells with low viability, which indicates cytotoxicity of this ND concentration manifesting after ~48 h of culturing. Changes in MP activation index were determined by the duration of culturing (duration of ND presence in MP) rather than ND concentration in the medium (Table 1). The capacity of MP to endocytose ND was related to ND capture by MP irrespective of the duration of ND presence in their vacuolar system; ND in concentrations of 30 and 60 μg/ml stimulated ROS production more actively after 24 and 48 h (Table 1). These data suggested that ND in concentrations of 10 to 20 μg/ml in the culture medium modulated MP functions not related to cytotoxicity, that is, exhibited biocompatibility. For this reason, we studied the expression of the key cytokines determining MP involvement in some typical pathological processes, in response to 48-h incubation with ND, added into culture medium in a concentration of 20 μg/ml. The percentage of MP expressing GM-CSF increased 2-fold, of MP expressing TGF-β increased 2.5 times (Table 2). The percentage of MP expressing IL-1α decreased by 39%, while the level of MP expressing TNF-α remained unchanged. The level of MP expressing bFGF decreased more than 2-fold.
93.80±5.84 95.9±4.3 94.80±4.07
20
30
60
98.20±2.36 96.10±2.37 95.70±3.54 66.10±2.64
10
20
30
60
ND, μg/ml
90.70±3.69
60 97.30±3.09
97.30±3.62
30
control
93.80±5.17 95.10±4.32
10
20
ND, μg/ml
97.40±3.72
60 97.50±4.32
96.60±4.56 97.90±3.65
20
30
94.90±4.87
10
control
ND, μg/ml
97.5±3.5
95.60±4.27
10
ND, μg/ml
control
93.50±5.54
control
Living MP, %
757.70±37.88+ 789.30±39.47+
93.30±6.68+ 95.80±4.46+
643.20±32.16 738.90±36.95
89.20±5.87
75.10±4.43
–
–
781.30±39.07+
91.30±7.25+
51.00±5.76+
75.30±5.65+
82.50±5.38
89.10±3.66
87.1±4.85*
79.60±3.86
92.90±3.26+
733.70±36.68+
79.60±6.37
985.50±64.05
1167.60±70.22
262.5±11.3+
243.30±10.38+
182.90±9.16 196.20±8.45+
1182.10±65.11
160.70±7.66
236.40±9.56+
214.80±9.14+
179.20±8.26
162.90±7.19
149.00±7.25
179.70±9.67
168.40±8.62+
154.1±7.9
139.30±6.41
124.20±6.88*
132.10±8.97
127.20±7.78
120.40±8.17
105.00±6.73
95.10±5.25
Index of ROS production by MP
1342.40±87.26+
967.50±58.05
1435.60±86.13+
1467.20±95.36+
1152.70±69.12 1491.50±89.55+
75.20±5.54
922.50±50.73*
867.70±52.02+
850.60±55.28+
826.60±53.73
+
697.50±41.85
675.80±37.12*
562.50±36.56
89.70±4.53+
620.20±31.12
65.6±4.1
91.80±3.57+
340.50±17.15+ –
86.40±4.45+
270.1±13.5+
76.60±4.16
+
180.60±9.08
71.70±2.56
+
62.70±3.55*
120.20±6.12
–
74.80±4.09+
40.50±2.03+
540.20±32.43
517.30±33.61
47.20±3.27 51.90±2.31+
495.1±29.7
+
472.50±25.98
Activation index of MP
+
17.00±0.85
42.00±3.49
23.1±2.9
Flattened MP, %
25.60±1.28+
0
–
Phagocytic activity index of MP
707.90±35.43
74.90±6.28
72.20±5.82*
–
45.50±5.96
44.20±4.97
42.60±3.91
39.10±3.59*
–
5.50±1.12
4.60±0.78
3.00±0.64
0
–
MP with phagocytic vacuoles, %
Note. Phagocytic activities for 3, 24, and 48 h periods were evaluated in comparison with the corresponding value for 1 h (control). p
500
Bulletin of Experimental Biology and Medicine, Vol. 158, No. 4, February, 2015
NANOTECHNOLOGIES In Vitro Effects of Nanosized Diamond Particles on Macrophages V. A. Shkurupy*,**, S. A. Arkhipov*,**, D. V. Neshchadim*, E. S. Akhramenko*, and A. V. Troitskii* Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 158, No. 10, pp. 503-506, October, 2014 Original article submitted January 18, 2014 The effects of synthetic diamond nanoparticles (4-6 nm) on mouse macrophage biotropism and biocompatibility and the modulation of the macrophage functions (expression of IL-1α, TNF-α, GM-CSF, bFGF, and TGF-β) by nanoparticles in different concentrations were studied in vitro during exposure of different duration. Macrophage endocytosis of nanodiamonds increased with increasing the concentration of nanoparticles in culture and incubation time. Nanodiamonds exhibited high biotropism and biocompatibility towards macrophages; in doses of 10-20 μg/ml, they induced expression of GM-CSF and TGF-β, inhibited expression of bFGF, and did not stimulate IL-1α and TNF-α. These data indicate that nanodiamond capture by macrophages in the studied experimental model led to modulation of the functional status of macrophages that determine their capacity to stimulate reparative processes without increasing proinflammatory and profibrogenic status. Key Words: nanodiamonds; macrophages; biocompatibility; cytokine expression; in vitro
Biocompatibility is one of the most important characteristics of nanosized artificial materials for medicine [3]. Artificial nanosized diamonds (ND) is a prospective nanomaterial [1,6,8,9]. However, the data on ND biotropism are insufficient for their wide use in pharmacy and medical practice because of their probable effects which can become risk factors in certain diseases [2,5,7,8]. Despite obvious chemical inertness, ND are not biologically inert. Being captured by macrophages, they inhibit expression of chemokine CCL2 and PDGF genes in these cells [10]. Macrophages capture many corpuscular objects and are involved in all pathological processes in mammals. We studied the effects of ND particles on the viability and some basic functions of macrophages in vitro. *Research Center of Clinical and Experimental Medicine, Siberian Division of the Russian Academy of Medical Sciences, Novosibirsk; **Novosibirsk State Medical University, Russia. Address for correspondence: [email protected]. S. A. Arkhipov
MATERIALS AND METHODS Two-month-old male BALB/c mice (21-22 g) from Breeding Center of Institute of Cytology and Genetics, Russian Academy of Science were used in the study. Peritoneal macrophages (MP) were isolated after the animals were sacrificed by cervical dislocation under ether narcosis [5]. The cells from peritoneal transsudate were pre-cultured (37oC, 3 h) on slides for MP adhesion (106 cells in 1.5 ml medium 199 with 10% fetal calf serum, FCS). Artificial ND (4-6 nm; UDA-V-GO brand; Research and Production Association “Altai”) were used in the study. Bioactivity of ND towards peritoneal MP was evaluated 1, 3, 24, and 48 h after ND addition to the primary 24-h MP cultures. Sterile suspension (1.6%) of ND in bidistilled water was added into culture medium (medium 199 with 10% FCS) to the final concentrations of 10, 20, 30, and 60 μg/ml. The suspension was then
0007-4888/15/15840500 © 2015 Springer Science+Business Media New York
V. A. Shkurupy, S. A. Arkhipov, et al.
thoroughly mixed and exposed to ultrasonic disintegration (MUZA-0.1/22-M device) for 10 sec at 75 W. The viability of MP (percentage of living cells) in cultures was evaluated by trypan blue staining [5]. Activation of MP was evaluated by activation index corresponding to the area (square pixels) of MP flattened after ND capturing. ROS production was indirectly evaluated by the NBT test [5]. The content of formazan formed in MP was evaluated in arbitrary units (stained area in square pixels) in the microphotographs made under an AxioVision Z1 microscope (Carl Zeiss) at ×400 using VideoTest-Morpho 3.2 software. ROS production by MP was evaluated by ROS production index, equal to the product of the area of MP formazan binary images and the percentage of formazan-containing MP (for respective cultures). Phagocytic activity of MP was evaluated by the phagocytic activity index equal to the product of the phagocytic number and the phagocytic index (percentage of MP with endocytic vacuoles) of the respective culture. ND particles were detected in MP endosomes using combined illumination of the preparations – in transmitting light and induced fluorescence simultaneously [5]. Immunohistochemical studies of MP were carried out by the indirect method. ND (20 μg/ml) were added into 24-h MP cultures according to the same protocol as in studies of the cytophysiological characteristics. Macrophages were cultured with ND for 48 h at 37oC. Macrophage expression of IL-1α, GM-CSF, TNF-α, bFGF, and TGF-β was detected using monoclonal antibodies: anti-mouse IL-1α (Hamster IgG1, λ; Becton Dickinson), anti-mouse GM-CSF (isotype: Rat IgG2a; clone MP1-22E9; Becton Dickinson); anti-mouse TNF-α (Rat IgG1; clone MP6-XT22; Becton Dickinson); anti-TGFbeta (polyclonal antibody, LS-B5663-50; LifeSpan BioSciences); anti-bFGF (polyclonal antibody, bs bs-0217R; Bioss). Rat first antibodies were visualized by anti-rat Ig HRP Detection Kit (Cat. #552013; BD Pharmingen). Rabbit first antibodies were visualized by ImmPRESS anti-rabbit Ig Polymer Detection Kit – Vector Labs MP-7401-15. Hamster first antibodies were visualized by biotinilated anti-hamster antibodies (Cat. #550335; BD Pharmingen) and streptavidin-HRP from Novocastra 250 immunocytochemistry kit. The results were statistically processed by variational statistical methods. The data were presented as the arithmetic means and errors in the means. The significance of differences in the means was evaluated by the nonparametric Mann–Whitney test. The results were statistically processed by Statistica 7.0 software (StatSoft Inc.).
RESULTS Culturing of peritoneal MP in a medium with ND particles in concentrations of 10-30 μg/ml caused no
501 changes in MP viability parameters irrespective of the duration of culturing (Table 1). The level of viable cells decreased by 33% after MP culturing in a medium with 60 μg/ml ND for 48 h. The percentage of MP with endocytic vacuoles containing ND increased with increasing the duration of culturing and ND concentration (Table 1). Endocytic activity index increased with increasing the duration of culturing and ND concentration in the medium; however, after 24 h of culturing it little depended on ND concentration and seemed to be determined by only endocytic capacity of the MP vacuolar system and their capacity to form endocytic vacuoles. Endocytic vacuoles containing ND often fused to form much larger endosomes (Table 1). The capacity of MP to flattening is an integral indicator of their activity associated with the cytoskeleton conformation during realization of the endocytic function and its “energy support” [4-6]. Capture of ND by MP was conjugated with an increase in the counts of flattened cells during the very first hour of their reactions with ND, and the dynamics of this process correlated with ND concentrations in culture medium. However, the involvement of MP in the flattening process was less dynamic after 3 h of culturing and until 48 h of culturing. The percentage of flattened MP decreased by 42% after 48 h of culturing in medium with 60 μg/ml ND, presumably because of a 33% decrease in the level of viable MP during this period (Table 1). Presumably, this was a pool of cells with low viability, which indicates cytotoxicity of this ND concentration manifesting after ~48 h of culturing. Changes in MP activation index were determined by the duration of culturing (duration of ND presence in MP) rather than ND concentration in the medium (Table 1). The capacity of MP to endocytose ND was related to ND capture by MP irrespective of the duration of ND presence in their vacuolar system; ND in concentrations of 30 and 60 μg/ml stimulated ROS production more actively after 24 and 48 h (Table 1). These data suggested that ND in concentrations of 10 to 20 μg/ml in the culture medium modulated MP functions not related to cytotoxicity, that is, exhibited biocompatibility. For this reason, we studied the expression of the key cytokines determining MP involvement in some typical pathological processes, in response to 48-h incubation with ND, added into culture medium in a concentration of 20 μg/ml. The percentage of MP expressing GM-CSF increased 2-fold, of MP expressing TGF-β increased 2.5 times (Table 2). The percentage of MP expressing IL-1α decreased by 39%, while the level of MP expressing TNF-α remained unchanged. The level of MP expressing bFGF decreased more than 2-fold.
93.80±5.84 95.9±4.3 94.80±4.07
20
30
60
98.20±2.36 96.10±2.37 95.70±3.54 66.10±2.64
10
20
30
60
ND, μg/ml
90.70±3.69
60 97.30±3.09
97.30±3.62
30
control
93.80±5.17 95.10±4.32
10
20
ND, μg/ml
97.40±3.72
60 97.50±4.32
96.60±4.56 97.90±3.65
20
30
94.90±4.87
10
control
ND, μg/ml
97.5±3.5
95.60±4.27
10
ND, μg/ml
control
93.50±5.54
control
Living MP, %
757.70±37.88+ 789.30±39.47+
93.30±6.68+ 95.80±4.46+
643.20±32.16 738.90±36.95
89.20±5.87
75.10±4.43
–
–
781.30±39.07+
91.30±7.25+
51.00±5.76+
75.30±5.65+
82.50±5.38
89.10±3.66
87.1±4.85*
79.60±3.86
92.90±3.26+
733.70±36.68+
79.60±6.37
985.50±64.05
1167.60±70.22
262.5±11.3+
243.30±10.38+
182.90±9.16 196.20±8.45+
1182.10±65.11
160.70±7.66
236.40±9.56+
214.80±9.14+
179.20±8.26
162.90±7.19
149.00±7.25
179.70±9.67
168.40±8.62+
154.1±7.9
139.30±6.41
124.20±6.88*
132.10±8.97
127.20±7.78
120.40±8.17
105.00±6.73
95.10±5.25
Index of ROS production by MP
1342.40±87.26+
967.50±58.05
1435.60±86.13+
1467.20±95.36+
1152.70±69.12 1491.50±89.55+
75.20±5.54
922.50±50.73*
867.70±52.02+
850.60±55.28+
826.60±53.73
+
697.50±41.85
675.80±37.12*
562.50±36.56
89.70±4.53+
620.20±31.12
65.6±4.1
91.80±3.57+
340.50±17.15+ –
86.40±4.45+
270.1±13.5+
76.60±4.16
+
180.60±9.08
71.70±2.56
+
62.70±3.55*
120.20±6.12
–
74.80±4.09+
40.50±2.03+
540.20±32.43
517.30±33.61
47.20±3.27 51.90±2.31+
495.1±29.7
+
472.50±25.98
Activation index of MP
+
17.00±0.85
42.00±3.49
23.1±2.9
Flattened MP, %
25.60±1.28+
0
–
Phagocytic activity index of MP
707.90±35.43
74.90±6.28
72.20±5.82*
–
45.50±5.96
44.20±4.97
42.60±3.91
39.10±3.59*
–
5.50±1.12
4.60±0.78
3.00±0.64
0
–
MP with phagocytic vacuoles, %
Note. Phagocytic activities for 3, 24, and 48 h periods were evaluated in comparison with the corresponding value for 1 h (control). p
