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OPEN
Received: 3 May 2017 Accepted: 13 July 2017 Published: xx xx xxxx
An Evaluation of the in vivo Safety of Nonporous Silica Nanoparticles: Ocular Topical Administration versus Oral Administration Martha Kim1, Joo-Hee Park 1, Hyejoong Jeong2, Jinkee Hong2, Woo Sung Choi3, Byung-Han Lee3 & Choul Yong Park1 Nonporous silica nanoparticles (SiNPs) have potential as promising carriers for ophthalmic drugs. However, the in vivo safety of ocular topical SiNPs remains unclear. This study investigated the in vivo safety of oral and ocular topical applications of 100 nm-sized SiNPs in Sprague-Dawley rats. The rats were divided into the following four groups: low-dose oral administration (total 100 mg/kg of SiNPs mixed with food for one week), high-dose oral administration (total 1000 mg/kg of SiNPs mixed with food for one week), ocular topical administration (10 mg/ml concentration, one drop, applied to the right eyes four times a day for one month), or a negative control (no SiNP treatment). The rats were observed for 12 weeks to investigate any signs of general or ocular toxicity. During the observation period, no differences were observed in the body weights, food and water intakes, behaviors and abnormal symptoms of the four groups. No animal deaths occurred. After 12 weeks, hematologic, blood biochemical parameters and ophthalmic examinations revealed no abnormal findings in any of the animals. The lack of toxicity of the SiNPs was further verified in autopsy findings of brain, liver, lung, spleen, heart, kidneys, intestine, eyeballs, and ovaries or testes. Recent advances in particle engineering technology have expanded the applications of nanoparticles (NPs), which can now serve as novel drug carriers. Nonporous silica NPs (SiNPs), which are commonly used as additives in cosmetics, printer toners, packaging, and imaging, represent promising drug carrier systems1. The stable chemical structure, large surface to volume ratios, ease of surface modification, and tolerable biodegradability of SiNPs greatly increase their attractiveness in biological applications as nanocarriers1. As is well known, ocular drug penetration is difficult due to extrinsic and intrinsic ocular barriers, such as the tear film, mucus barrier, tight junction of the corneal epithelium, hydrophilicity of the corneal epithelium, and hydrophobicity of the corneal stroma2. Therefore, nano-based drug carrier systems, such as SiNPs, are a promising field of research for efficient ocular drug delivery3, 4. Several recent reports described successful intraocular drug or gene delivery via various NPs, further increasing expectations regarding their potential5–11. Despite the potential of NPs, such as SiNPs, a major concern is their safety in medical applications12, 13. Research has shown that inhalation of some SiNPs can damage pulmonary cells via the induction of oxidative stress14. Disturbance of calcium homeostasis of neuronal cells after SiNP exposure has also been previously reported15. Concerns about the intestinal toxicity and genotoxicity of SiNPs have also been raised16. Nevertheless, recent research pointed to the safety of SiNPs. For example, Ryu et al.17 reported that SiNPs had no toxic effects, including no such effects on internal organs, after dermal topical application of SiNPs for 90 days. In another report, SiNPs elicited only minimal biological toxicity in intestinal cells compared to marked toxicity caused by zinc oxide NPs18. Previously, our group reported the in vitro safety of SiNPs on cultured human corneal epithelial cells and keratocytes19, 20. In our previous study, various sized SiNPs (50, 100, and 150 nm) did not result in any significant impairment of cellular viability after exposure for 24 h to concentrations up to 100 μg/ml. It is likely that the nanotoxicity of SiNPs are cell-type dependent13. Different types of cells are exposed to SiNPs, depending 1
Department of Ophthalmology, Dongguk University, Ilsan Hospital, Goyang, South Korea. 2School of Chemical Engineering and Material Science, Chung-Ang University, Seoul, South Korea. 3Laboratory Animal Center, Osong Medical Innovation Foundation, Cheongju, South Korea. Correspondence and requests for materials should be addressed to C.Y.P. (email: [email protected]) SCIEntIFIC REPOrTS | 7: 8238 | DOI:10.1038/s41598-017-08843-9
1
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Group
Route
Number of animals
Dose
5 males and 5 females
G1 (Control) G2
oral
100 mg/kg
5 males and 5 females
G3
oral
1000 mg/kg
5 males and 5 females
G4
topical
0.1 ml q.i.d for 1 month
5 males and 5 females
Table 1. Groups, route of administration and dose levels of silica nanoparticles.
0 week G1
G2
G3
G4
1 week
2 week
3 week
4 week
5 week
6 week
7 week
8 week
9 week
10 week
11 week
12 week
Mean
205.9
234.9
255.3
273.7
287.1
288.9
301.7
312.9
323.3
314.7
328.1
333.7
337.1
SD
38.5
45.5
56.0
63.5
67.9
70.9
71.4
75.1
87.4
78.8
83.9
86.7
89.0
N
10
10
10
10
10
10
10
10
10
10
10
10
10
Mean
204.6
241.0
262.6
281.7
297.8
301.6
310.7
322.8
332.7
346.3
345.0
347.0
351.0
SD
39.2
56.6
65.0
75.1
81.5
83.8
85.7
90.4
91.8
103.9
96.4
99.2
103.7
N
10
10
10
10
10
10
10
10
10
10
10
10
10
Mean
204.9
235.8
261.0
276.6
293.9
296.2
311.4
320.4
327.0
328.9
339.2
345.0
347.7
SD
39.1
50.3
59.2
70.4
76.1
79.3
78.9
86.9
89.5
92.1
97.7
99.4
103.8
N
10
10
10
10
10
10
10
10
10
10
10
10
10
Mean
205.2
228.7
243.9
263.2
277.3
287.9
306.0
314.6
327.7
323.1
331.9
337.4
342.7
SD
38.9
46.3
50.1
54.4
63.1
65.9
65.5
75.4
77.5
81.0
83.3
87.9
89.3
N
10
10
10
10
10
10
10
10
10
10
10
10
10
Table 2. Body weight (gram) changes of rats after SiNPs application up to 12 weeks. G: group, SD: standard deviation, N: number.
on the route of SiNP administration. Therefore, independent evaluations of the nanotoxicity of ocular topical administration of SiNPs are necessary to resolve whether SiNPs are safe for ophthalmic uses. The current study investigated the in vivo effects of oral and ocular topical SiNPs in rats over a 12-week period. General toxicity-related effects (e.g., changes in body weight and food and water intake) of the SiNPs, as well as specific effects on hematologic parameters, biochemical parameters, and vital organs, including eyeballs, were studied.
Results
Clinical observations. The rats were divided into four groups (n = 10 each): control (Group 1), low-dose oral intake (Group 2), high-dose oral intake (Group 3), and topical administration (Group 4). Each group contained an equal number of males and females (Table 1). Body weight, food and water intake, behaviors and abnormal symptoms (such as hyper/hypo-activity, abnormal breathing, abnormal locomotion, lethargy, paresis, paralysis, tremor, ruffled fur and eye discharge) were observed over a 12-week period after the administration of the SiNPs. No deaths occurred in any of the animal groups. As compared with the untreated controls, none of the groups showed body weight loss, differences in food and water intake, abnormal behaviors, or general abnormal symptoms (Table 2) (Fig. 1). Hematologic and biochemical examinations. The results of the hematologic and biochemical exami-
nations 12 weeks after the application of the SiNPs are shown in Tables 1,2 and 3. There were no differences in the hematologic parameters of the oral intake groups (Groups 2 and 3). However, as compared to the control group (Group 1), there was a significant decrease in the mean platelet volume in Group 2. There were no differences in the hematologic parameters of the topical administration group (Group 4) compared to those of the oral intake groups. However, the mean platelet count (P
OPEN
Received: 3 May 2017 Accepted: 13 July 2017 Published: xx xx xxxx
An Evaluation of the in vivo Safety of Nonporous Silica Nanoparticles: Ocular Topical Administration versus Oral Administration Martha Kim1, Joo-Hee Park 1, Hyejoong Jeong2, Jinkee Hong2, Woo Sung Choi3, Byung-Han Lee3 & Choul Yong Park1 Nonporous silica nanoparticles (SiNPs) have potential as promising carriers for ophthalmic drugs. However, the in vivo safety of ocular topical SiNPs remains unclear. This study investigated the in vivo safety of oral and ocular topical applications of 100 nm-sized SiNPs in Sprague-Dawley rats. The rats were divided into the following four groups: low-dose oral administration (total 100 mg/kg of SiNPs mixed with food for one week), high-dose oral administration (total 1000 mg/kg of SiNPs mixed with food for one week), ocular topical administration (10 mg/ml concentration, one drop, applied to the right eyes four times a day for one month), or a negative control (no SiNP treatment). The rats were observed for 12 weeks to investigate any signs of general or ocular toxicity. During the observation period, no differences were observed in the body weights, food and water intakes, behaviors and abnormal symptoms of the four groups. No animal deaths occurred. After 12 weeks, hematologic, blood biochemical parameters and ophthalmic examinations revealed no abnormal findings in any of the animals. The lack of toxicity of the SiNPs was further verified in autopsy findings of brain, liver, lung, spleen, heart, kidneys, intestine, eyeballs, and ovaries or testes. Recent advances in particle engineering technology have expanded the applications of nanoparticles (NPs), which can now serve as novel drug carriers. Nonporous silica NPs (SiNPs), which are commonly used as additives in cosmetics, printer toners, packaging, and imaging, represent promising drug carrier systems1. The stable chemical structure, large surface to volume ratios, ease of surface modification, and tolerable biodegradability of SiNPs greatly increase their attractiveness in biological applications as nanocarriers1. As is well known, ocular drug penetration is difficult due to extrinsic and intrinsic ocular barriers, such as the tear film, mucus barrier, tight junction of the corneal epithelium, hydrophilicity of the corneal epithelium, and hydrophobicity of the corneal stroma2. Therefore, nano-based drug carrier systems, such as SiNPs, are a promising field of research for efficient ocular drug delivery3, 4. Several recent reports described successful intraocular drug or gene delivery via various NPs, further increasing expectations regarding their potential5–11. Despite the potential of NPs, such as SiNPs, a major concern is their safety in medical applications12, 13. Research has shown that inhalation of some SiNPs can damage pulmonary cells via the induction of oxidative stress14. Disturbance of calcium homeostasis of neuronal cells after SiNP exposure has also been previously reported15. Concerns about the intestinal toxicity and genotoxicity of SiNPs have also been raised16. Nevertheless, recent research pointed to the safety of SiNPs. For example, Ryu et al.17 reported that SiNPs had no toxic effects, including no such effects on internal organs, after dermal topical application of SiNPs for 90 days. In another report, SiNPs elicited only minimal biological toxicity in intestinal cells compared to marked toxicity caused by zinc oxide NPs18. Previously, our group reported the in vitro safety of SiNPs on cultured human corneal epithelial cells and keratocytes19, 20. In our previous study, various sized SiNPs (50, 100, and 150 nm) did not result in any significant impairment of cellular viability after exposure for 24 h to concentrations up to 100 μg/ml. It is likely that the nanotoxicity of SiNPs are cell-type dependent13. Different types of cells are exposed to SiNPs, depending 1
Department of Ophthalmology, Dongguk University, Ilsan Hospital, Goyang, South Korea. 2School of Chemical Engineering and Material Science, Chung-Ang University, Seoul, South Korea. 3Laboratory Animal Center, Osong Medical Innovation Foundation, Cheongju, South Korea. Correspondence and requests for materials should be addressed to C.Y.P. (email: [email protected]) SCIEntIFIC REPOrTS | 7: 8238 | DOI:10.1038/s41598-017-08843-9
1
www.nature.com/scientificreports/
Group
Route
Number of animals
Dose
5 males and 5 females
G1 (Control) G2
oral
100 mg/kg
5 males and 5 females
G3
oral
1000 mg/kg
5 males and 5 females
G4
topical
0.1 ml q.i.d for 1 month
5 males and 5 females
Table 1. Groups, route of administration and dose levels of silica nanoparticles.
0 week G1
G2
G3
G4
1 week
2 week
3 week
4 week
5 week
6 week
7 week
8 week
9 week
10 week
11 week
12 week
Mean
205.9
234.9
255.3
273.7
287.1
288.9
301.7
312.9
323.3
314.7
328.1
333.7
337.1
SD
38.5
45.5
56.0
63.5
67.9
70.9
71.4
75.1
87.4
78.8
83.9
86.7
89.0
N
10
10
10
10
10
10
10
10
10
10
10
10
10
Mean
204.6
241.0
262.6
281.7
297.8
301.6
310.7
322.8
332.7
346.3
345.0
347.0
351.0
SD
39.2
56.6
65.0
75.1
81.5
83.8
85.7
90.4
91.8
103.9
96.4
99.2
103.7
N
10
10
10
10
10
10
10
10
10
10
10
10
10
Mean
204.9
235.8
261.0
276.6
293.9
296.2
311.4
320.4
327.0
328.9
339.2
345.0
347.7
SD
39.1
50.3
59.2
70.4
76.1
79.3
78.9
86.9
89.5
92.1
97.7
99.4
103.8
N
10
10
10
10
10
10
10
10
10
10
10
10
10
Mean
205.2
228.7
243.9
263.2
277.3
287.9
306.0
314.6
327.7
323.1
331.9
337.4
342.7
SD
38.9
46.3
50.1
54.4
63.1
65.9
65.5
75.4
77.5
81.0
83.3
87.9
89.3
N
10
10
10
10
10
10
10
10
10
10
10
10
10
Table 2. Body weight (gram) changes of rats after SiNPs application up to 12 weeks. G: group, SD: standard deviation, N: number.
on the route of SiNP administration. Therefore, independent evaluations of the nanotoxicity of ocular topical administration of SiNPs are necessary to resolve whether SiNPs are safe for ophthalmic uses. The current study investigated the in vivo effects of oral and ocular topical SiNPs in rats over a 12-week period. General toxicity-related effects (e.g., changes in body weight and food and water intake) of the SiNPs, as well as specific effects on hematologic parameters, biochemical parameters, and vital organs, including eyeballs, were studied.
Results
Clinical observations. The rats were divided into four groups (n = 10 each): control (Group 1), low-dose oral intake (Group 2), high-dose oral intake (Group 3), and topical administration (Group 4). Each group contained an equal number of males and females (Table 1). Body weight, food and water intake, behaviors and abnormal symptoms (such as hyper/hypo-activity, abnormal breathing, abnormal locomotion, lethargy, paresis, paralysis, tremor, ruffled fur and eye discharge) were observed over a 12-week period after the administration of the SiNPs. No deaths occurred in any of the animal groups. As compared with the untreated controls, none of the groups showed body weight loss, differences in food and water intake, abnormal behaviors, or general abnormal symptoms (Table 2) (Fig. 1). Hematologic and biochemical examinations. The results of the hematologic and biochemical exami-
nations 12 weeks after the application of the SiNPs are shown in Tables 1,2 and 3. There were no differences in the hematologic parameters of the oral intake groups (Groups 2 and 3). However, as compared to the control group (Group 1), there was a significant decrease in the mean platelet volume in Group 2. There were no differences in the hematologic parameters of the topical administration group (Group 4) compared to those of the oral intake groups. However, the mean platelet count (P
