Subscriber access provided by WEBSTER UNIV
Article
Fate of Carbon Nanotubes Locally Implanted in Mice Evaluated by NearInfrared Fluorescence Imaging: Implications for Tissue Regeneration Eri Hirata, Masako Yudasaka, Natsumi Ushijima, Norihito Sakaguchi, Yukari Maeda, Takeshi Tanaka, Hiromichi Kataura, and Atsuro Yokoyama ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b02267 • Publication Date (Web): 04 Feb 2019 Downloaded from http://pubs.acs.org on February 4, 2019
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 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 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.
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 22 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
ACS Applied Nano Materials
Fate of Carbon Nanotubes Locally Implanted in
Mice
Evaluated
Fluorescence
Imaging:
by
Near-Infrared
Implications
for
Tissue Regeneration Eri Hirata,†* Masako Yudasaka,§ Natsumi Ushijima,† Norihito Sakaguchi,ǁ Yukari Maeda,† Takeshi Tanaka,§ Hiromichi Kataura,§ and Atsuro Yokoyama† †Graduate
School of Dental Medicine, Hokkaido University, Sapporo 060-8586, Japan Research Institute (NMRI), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central, Tsukuba 305-8565 Japan ǁCenter for Advanced Research of Energy Conversion Materials, Hokkaido University, Sapporo 060-8628, Japan §Nanomaterials
Corresponding author. Tel: +81-11-706-4270 Fax: +81-11-706-4903 E-mail address:
[email protected] *(E. Hirata)
1 ACS Paragon Plus Environment
ACS Applied Nano Materials 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
Abstract Carbon nanomaterials (CNMs) are used in various functional materials and products as well as in basic research, mainly in engineering. Because of their high biocompatibility, use of CNMs in bioapplications is widely anticipated. Many researchers have examined the kinetics and toxicity of intravenously administered CNMs. However, little research on the in vivo kinetics of locally implanted CNMs has been reported. In this study, single-walled carbon nanotubes (SWCNTs) were implanted between the periosteum and parietal bone of mice. The in vivo kinetics of the implanted SWCNTs were observed by near-infrared (NIR) fluorescence imaging (excitation wavelength, 730 nm; emission wavelength, >1000 nm) because the body is highly transparent to light in the NIR region and autofluorescence in this region is low, making NIR fluorescence suitable for bioimaging. SWCNTs were visible in the subcutaneous tissue and medullary cavity. Fluorescence was observed in the cranial region, and its intensity gradually decreased over 24 h. Fluorescence was not observed in other organs, including the liver, spleen, and lung, in wholebody imaging experiments. After 56 d, fluorescence weakened, but was still clearly observed in the cranial region. The weakening of fluorescence over time was possibly because SWCNTs became bundled or adsorbed fluorescence-quenching materials in the tissue. These findings suggest that locally implanted SWCNTs remain at the site of implantation and do not accumulate in detectable quantities in other organs. We expect that this proof-of-concept for the biodistribution of SWCNTs after local implantation will promote future studies on the application of CNMs as biomaterials. Keywords Carbon nanotube, near-infrared fluorescence imaging, biodistribution, local implantation, biomaterials, transmission electron microscopy observation
2 ACS Paragon Plus Environment
Page 2 of 22
Page 3 of 22 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
ACS Applied Nano Materials
Introduction Carbon nanotubes (CNTs) are used in various functional materials and products, predominantly in engineering but also in basic research. The high biocompatibility of CNTs makes them attractive for use in bioapplications.1–6 We previously reported that multi-walled CNT (MWCNT)-coated substrates can be effective for adhesion and differentiation of osteoblasts and MWCNT-coated collagen sponges have a favorable biocompatibility profile for bone.7–9 Singlewalled CNT (SWCNT)–collagen hybrid hydrogels have been investigated as functional scaffold materials for cardiac construct engineering10 and conductive biomaterials for nerve repair.11 These biomaterials were implanted locally to promote tissue regeneration. Biodistribution is an important factor of in vivo behavior when evaluating the biosafety of nanomaterials by analytical methods. Many researchers have performed intravenous administration of SWCNTs to examine their kinetics and toxicity.12–14 However, little research on the in vivo kinetics of locally implanted SWCNTs has been reported. SWCNTs are promising as probes because of their excellent intrinsic optical properties such as fluorescence, Raman, and absorption spectral responses.15–17 Probes emitting near-infrared (NIR) fluorescence in the longwavelength region (1100–1400 nm; NIR-II) are expected to be suitable for bioimaging because they show deep penetration and low scattering in cells18,19 and the living body.20–24 For instance, Iverson et al.25 reported that alginate and PEG gels containing SWCNTs with a concentration of 10 mg L−1 displayed an optical detection limit of 5.4 mm at a depth of 5.1 mm in tissue under 785-nm laser excitation at 80 mW and 30 s of exposure. The body is highly transparent to light in the NIR region and the autofluorescence level in this region is low, meaning that NIR fluorescence is suitable for bioimaging. SWCNTs were visible in the subcutaneous tissue and medullary cavity.24 In the present study, SWCNTs are implanted between the periosteum and parietal bone of mice and their in vivo kinetics are evaluated by NIR fluorescence imaging and transmission election microscopy (TEM) observation.
3 ACS Paragon Plus Environment
ACS Applied Nano Materials 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
Results and Discussion Figure 1(a–g) show a time course of NIR fluorescence images in the cranial region where PLPEGSWCNT-gel
(PLPEG
is
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-
[amino(poly(ethylene-glycol))-5000] and gel is collagen gel) was implanted in the pocket made between the parietal bone and periosteum. The average fluorescence intensities in regions of interest (measuring 7 × 7 mm at the brightest area) of the images are plotted in Figure 1h. There are significant statistical differences between the groups with and without implanted PLPEGSWCNTs. The fluorescence intensity gradually weakened over 24 h. After 6 d, the fluorescence still remained observable at the implanted area, although it was weak. A possible reason for the decrease in fluorescence intensity may be flow of the PLPEG-SWCNTs from the implanted site to other areas. However, this is not considered plausible, as discussed below.
Figure 1. (a–g) Time course of NIR fluorescence images in the cranial region after implantation of PLPEG-SWCNTs (a, 1 h; b, 3 h; c, 5 h; d, 24 h; e, 6 d; f, 28 d; g, 56 d) and fluorescence intensities (h). In (h), the fluorescence intensities in the region with collagen gel are shown in black and those in the region implanted with PLPEG-SWCNT-gel are shown in red. CNT group: 1 h, n=5; 3 h, n=5; 5 h, n=5; 24 h, n=15; 6 d, n=10; 28 d, n=10; 56 d, n=5. Control group: 1 h, n=5; 24 h, n=5; 6 d, n=4; 28 d, n=4; 56 d, n=3. Asterisks indicate a statistically significant difference between two groups (p**
