Effects of Polydopamine Functionalization on Boron Nitride Nanotube

Subscriber access provided by Stockholm University Library

Article

The effects of Polydopamine Functionalization on Boron Nitride Nanotube Dispersion and Cytocompatibility. Marc A. Fernandez-Yague, Aitor Larrañaga, Olga Gladkovskaya, Alanna Stanley, Ghazal Tadayyon, Yina Guo, Jose-Ramon Sarasua, Syed A. M. Tofail, Dimitrios I Zeugolis, Abhay Pandit, and Manus Biggs Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00257 • Publication Date (Web): 17 Aug 2015 Downloaded from http://pubs.acs.org on August 25, 2015

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.

Bioconjugate Chemistry 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 43

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

Bioconjugate Chemistry

The effects of Polydopamine Functionalization on Boron Nitride Nanotube Dispersion and Cytocompatibility Marc A. Fernandez-Yague 1, Aitor Larrañaga 1,2, Olga Gladkovskaya 1, Alanna Stanley 3, Ghazal Tadayyon 1, Yina Guo 4, Jose-Ramon Sarasua 2, Syed A. M. Tofail 4, Dimitrios I. Zeugolis 1,5, Abhay Pandit 1, Manus J. Biggs 1. 1

Centre For Research in Medical Devices (CURAM), National University of Ireland Galway (NUIG), Galway, Ireland 2 School of Engineering, University of the Basque Country (UPV/EHU), Department of Mining-Metallurgy Engineering and Materials Science & POLYMAT, Bilbao, Spain 3 Department of Anatomy, National University of Ireland Galway (NUIG), Galway, Ireland 4 Department of Physics and Energy, and Materials and Surface Science Institute (MSSI), University of Limerick, Ireland. 5 Regenerative Modular & Developmental Engineering Laboratory (REMODEL), National University of Ireland Galway (NUI Galway), Galway, Ireland

Table of Contents

GRAPHICAL ABSTRACT ...................................................................................................................................................1 ABSTRACT ............................................................................................................................................................................1 INTRODUCTION ..................................................................................................................................................................2 RESULTS ................................................................................................................................................................................4 BNNT DISPERSION IN TRIS-HCL BUFFER .................................................................................................................4 FIGURE 1 ...............................................................................................................................................................5 CHARACTERISATION OF PD-FUNCTIONALIZED BNNTS ......................................................................................5 FIGURE 2 ...............................................................................................................................................................6 FIGURE 3 ...............................................................................................................................................................7 FIGURE 4 ...............................................................................................................................................................8 FIGURE 5 ...............................................................................................................................................................9 CYTOTOXICITY OF BNNTS ...........................................................................................................................................10 FIGURE 6 .............................................................................................................................................................10 FIGURE 7 .............................................................................................................................................................11 FIGURE 8 .............................................................................................................................................................12 FIGURE 9 .............................................................................................................................................................13 FIGURE 10 ...........................................................................................................................................................13 DISCUSSION .......................................................................................................................................................................13 FIGURE 11 ...........................................................................................................................................................16 CONCLUSION .....................................................................................................................................................................21 MATERIALS AND METHODS.........................................................................................................................................22

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

MATERIALS ........................................................................................................................................................................22 METHODS ...........................................................................................................................................................................22 COATING OF BNNTS WITH PD22 CHARACTERISATON OF BNNTS ................................................................................................................................. 23 TRANSMISSION ELECTRON MICROSCOPY (TEM) ................................................................................23 X-RAY PHOTOELECTRON SPECTROSCOPY (XPS) ................................................................................24 UV-VISIBLE SPECTROSCOPY ...................................................................................................................... 24

ATTENUATED TOTAL REFLECTION-INFRARED RED (ATR-IR) ................................................ 25 CELL CULTURE AND CYTOCOMPATIBILITY ASSAYS .................................................................. 25 ATTENUATED TOTAL REFLECTION-INFRARED RED (ATR-IR) ................................................ 25 LIVE/DEAD (CELL VIABILITY) ................................................................................................................ 25 DOUBLE STRANDED-DNA (DS-DNA) QUANTIFICATION ............................................................... 26 ALAMARBLUE® ........................................................................................................................................... 26 BIOLOGICAL TEM SAMPLE PREPARATION AND IMAGE ACQUISITION .............................. 26 ACKNOWLEDGMENTS ................................................................................................................................................ 27 REFERENCES .................................................................................................................................................................. 28


Page 2 of 43

Page 3 of 43

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


BNNT

2 nm

PD-BNNT

2 nm 20 nm

300 nm

Abstract Boron nitride nanotubes (BNNT) have unique physical properties, of value in biomedical applications; however, their dispersion and functionalization represent a critical aspect for their successful employment as biomaterials. In the present study, we report a process for the efficient disentangled BNNTs via a dual surfactant/polydopamine (PD) process. Highresolution transmission electron microscopy (HR-TEM) indicated that individual BNNT become coated with a uniform PD nanocoating, which significantly enhanced dispersion of BNNT in aqueous solutions. Furthermore, the cytocompatiblity of PD-coated BNNT was assessed in vitro with cultured human osteoblasts (HOBs) at concentrations of 1, 10 and 30



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 4 of 43

µg/ml and over three time-points (24, 48 and 72 hours). In this study it was demonstrated that PD-functionalized BNNTs become individually localized within the cytoplasm by endosomal escape and that concentrations of up to 30 ug/ml of PD-BNNTs were cytocompatible in HOBs cells up to after 72 hours of exposure. Key words: boron nitride nanotubes (BNNTs), human osteoblasts (HOBs), piezoelectric, polydopamine (PD), transmission electron microscopy (TEM) Introduction The discovery of nanoscale carbon formulations encompassing nanotube and twodimensional graphene has resulted in significant advances in the fields of biomaterials, and nanoelectronics. Boron nitride nanotubes (BNNT) like carbon nanotubes (CNT) have potential applications in both biomedical and non-conventional electronics thanks to their singular structural and physical properties 1-4. Although CNTs have been largely studied as tubular nanostructure for a number of different applications explored as next generation materials

7-9

5, 6

, BNNT are still being

. BNNT possess a similar physical structure to

CNTs, yet possess significantly different physicochemical properties. In particular, despite the remarkable mechanical properties of CNTs, recent studies have demonstrated that BNNTs possess significantly greater shear strength than CNTs 10, moreover, they have been shown to present higher thermal conductivity, superior electrical band gap properties and are more resistant to oxidation at high temperatures 11. As with, CNTs, in order to explore BNNTs as next generation materials it is crucial to develop specific methodologies for their correct dispersion and functionalization

12

. The

high insolubility of BNNT in water and organic solvents has proved problematic for the


Page 5 of 43

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


generation of BNNT formulations and nanocomposites and early studies have concentrated on identifying suitable methodologies to address BNNT dispersion

13-16

. Initial approaches

have focused on the same covalent and non-covalent methodologies used for CNT dispersion

17

. Specifically, acid treatment has been applied to cut and shorten CNTs;

however, this treatment produces irreversible structural changes and decrements in electronic and mechanical properties of the nanotubes

18-21

. Alternatively, non-covalent

approaches such the use of amphiphilic molecules for dispersion has been successfully applied for CNT formulations

22-26

commonly in conjunction with an ultra-sonication

process 27-33. In the present work, functionalization of BNNTs with a polydopamine chemistry (PD) was explored as a strategy to improve the dispersion of BNNTs in aqueous solutions and to simultaneously provide functional groups that can act as anchorage points for further incorporation of molecules with biological activity, such as proteins or peptides 34, 35. PD is a dopamine derived synthetic eumelanin polymer that contains both catechol and amine functionalities in its backbone 36, 37. Via simple immersion of substrates in a dilute aqueous solution of dopamine hydrochloride, a thin PD layer was deposited on the surface of the material. Those aromatic molecules of dopamine strongly interact with boron nitride through π-π stacking forces and van der Waals interactions. This approach has been explored as a strategy for dispersing many different micro- and nanoscale materials previously, including BNNTs and boron nitride platelets

38, 39

. The results of these studies

demonstrated improved dispersion in water and the generation of composite nanomaterials with enhanced mechanical and thermal properties 40, 41.



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 6 of 43

Interestingly, BNNTs possess a unique capacity of generating electric fields under mechanical stimulation (piezoelectricity)

12

, and recent studies indicate that the

piezoelectric response of BNNTs is higher than piezoelectric polymers such as PVDF 42-45. In order to take advantage of these properties, BNNT have been explored as a nanovector for intracellular release of drugs or for the delivery of electrical stimuli

42

; however, a

greater understanding of BNNT cytocompatibility is critical to facilitate further investigation in this area. The production of BNNT by different methodologies such as CBD or ball-milling results in the synthesis of BNNTs with large diameters (30-50 nm) and nanotube formulations with a significant persistence of metallic impurities. Conversely, the BNNTs employed in this study were synthesized using a high temperature pressurized vapor/condenser method (PVC). The BNNT produced possess a number of walls between 1 to 5, a high aspect ratio (300 m2/g), high crystallinity and relatively small diameters (2-5 nm). Recent studies exploring BNNT cytocompatibility have concluded that the high hydrophobicity of the BNNTs resulting from the high polarity of the bond B-N leads to a lower cytocompatibility than that of CNTs

46

. However, the degree of dispersion,

incorporation of biocompatible coatings and control over nanotube dimensions have yet to be explored as mediators of cytocompatibility. Considering BNNT cytocompatibility, PD is presented as a promising methodology to coat BNNTs for the generation of novel biomaterial formulations. Herein, we present the first study into the cytocompatibility of PD coated highly crystalline, small diameter BNNTs produced by catalyst-free high pressure and high temperature method 11.


Page 7 of 43

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 BNNT Dispersion in Tris-HCl Buffer Figure 1 shows representative images illustrating the process of BNNT PD coating and dispersion in a Tris-HCl solution. BNNTs were originally in the form of a dry sponge (Figure 1A). With the aid of an ionic surfactant - Sodium Dodecyl Benzene Sulphonate (SDBS), the dispersion became homogeneous and white colored after a 3 hour ultrasonication process, indicating the efficacy of SDBS for dispersing and de-bundling BNNT fibrils (Figure 1B). This dispersion was subsequently centrifuged at 5000 rpm at 4ºC for 5 min and the BNNT supernatant was collected for PD coating. After the dopamine selfpolymerization reaction, the dispersion was homogeneous and the color turned from white to brown (Figure 1C).



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

Figure 1. Schematic of the BNNT dispersion and PD-functionalization process. Pristine BNNTs were dispersed in a Tris-HCL under probe ultrasonication to yield a 40 mg/ml suspension (A). BNNT suspension was subsequently centrifuged at 5000 rpm and the supernatant retained (B). The BNNTs were incubated with dopamine hydrochloride under agitation for 18 h before centrifugation at 13,000 rpm and washing (C). Pristine BNNTs retain a fibrous sponge-like morphology in aqueous environments following a 3 hours sonication process (D). SDBS (0.5 mg/ml) facilitate dispersion of the BNNTs (E) which become


Page 8 of 43

Page 9 of 43

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


functionalized with a stable PD nanocoating following the addition of dopamine hydrochloride (1mg/ml) for 18 hours (F).

Characterization of PD-Functionalized BNNTs Low magnification TEM imaging was performed to assess BNNT dispersion before and after PD functionalization. Specifically, it was observed that uncoated BNNT suspensions tended to agglomerate into bundles of 4 to 6 microns in diameter (Figure 2A). In contrast, PD coated-BNNTs were well dispersed and when cast onto a surface formed a percolated network (Figure 2B).

Figure 2. Low magnification TEM images of non-functionalized and PD-functionalized BNNTs cast on lacey carbon grids. SDBS dispersed suspensions of BNNTS were undispersed and formed micro-aggregates in suspension (A) PD-functionalized BNNTs were observed to undergo a complete disentanglement, resulting in significantly enhanced dispersion (B). This was further confirmed via fluorescent imaging of peroxide activated autofluorescent BNNTs (insert). Insert scale bar, 1 µm.



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

BNNT were observed to possess well-defined walls, ranging in number from 4-6. Moreover, the unaltered hexagonal arrangements of B and N atoms were observable in pristine BNNTs (Figure 3A). The presence of an investing PD coating was confirmed by Energy-dispersive X-ray spectroscopy (EDS) analysis and high-resolution TEM imaging. TEM analysis also allowed the direct observation of PD functionalization of BNNTs, where it was possible to identify a homogenous 1.5 nm thick coating along the nanotube surface (Figure 3B & C). The presence of a superficial PD layer was further confirmed via dynamic light scattering (DLS) and it was noted that following PD functionalization the mean hydrodynamic radius was increased from 228±3 to 257±4 nm (see supplementary information S1 – S3). EDS analysis of SDBS dispersed indicated B and N peaks of similar intensity, and also the presence of SDBS as indicated by the Na and S signals. In contrast, following PD functionalization, Na and S signals were reduced and an increased C signal was obtained (Figure 3D & E).


Page 10 of 43

Page 11 of 43

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


Figure 3. High-resolution TEM and EDS of non-functionalized and PD-functionalized BNNTs. SDBS dispersed BNNTs were observed to possess multiple well-defined walls and a hexagonal atomic arrangement. (A). Conversely, PD-functionalized BNNTs possessed an amorphous surface coating (B). Ultra-high magnification (1.5 M) of this coating indicated the thickness of to be approximately 1.5 nm (C). Elemental analysis confirmed levels of Boron Nitrogen and Sodium in SDBS dispersed BNNTs (D) Conversely, boron, nitrogen and sodium were reduced in PD-functionalized BNNTs and the presence of carbon increased (E).

The presence of PD coating was also confirmed by X-ray photoelectron spectroscopy (XPS) that indicated significant differences in elemental composition between BNNTs and PD -BNNTs (Figure 4). Specifically, the carbon content (as indicated by the C 1s peak) increased from 4.6% for pristine BNNTs to 16.9 % for SDBS dispersed BNNTs and to 24.9% for PD-functionalized BNNTs.



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

Figure 4. XPS survey spectra and element quantification of pristine, SDBS dispersed, and PD-functionalized BNNTs. SDBS dispersion significantly increased the content of carbon - C1 s (16.9%) and decreased the boron - B 1s (33.2%) and nitrogen - N 1s (43.9%) relative to pristine BNNTs. A further significant increase in the carbon content (24.9%) was observed in PD-functionalized BNNTs attributed to the carbon and nitrogen backbone of the PD. Sulfur content (S 2p), originating from SDBS immersion was present only on SDBSdispersed (0.7%) and PD-Functionalized BNNTs (0.4%).

To quantify the significant effects of PD functionalization on BNNT dispersion the absorbance profiles of BNNT, PD and PD-functionalized BNNTs as a function of the wavelength was quantified (Figure 5). An increase in the absorbance for PD-functionalized BNNT with respect to SDBS dispersed BNNT (non-functionalized) was observed. This


Page 12 of 43

Page 13 of 43

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


increase in adsorption was also observed as a function of concentration. Here we assessed absorbance with concentrations ranging from 0 – 0.8 mg. In this manner, it was possible to estimate the final concentration of PD in a PD-functionalized BNNT dispersion following centrifugation. Accordingly, a value around 20 µg/ml was obtained by assessing the absorbance of SDBS dispersed PD (see supplementary information S4) and the aid of equation 1. The effects of SDBS coating were further evaluated via UV-VIS (see supplementary information S5).

Figure 5. Absorbance spectroscopy of non-functionalized BNNT, PD-functionalized BNNT and PD (0.5 mg/ml) (A) and standard curves showing the extinction coefficients (ki) for non-functionalized BNNT (B) and Dopamine (C). The correlation of the curve was 99.8% for n= 3 samples.



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

Cytotoxicity of BNNTs Osteoblasts were cultured in complete media containing SDBS dispersed and PDfunctionalized BNNT’s of increasing concentrations. Figures 6 and 7 show representative fluorescent microscopy images of live/dead labeled cells corresponding to BNNT concentrations of 1, 10 and 30 µg/ml acquired after 24, 48 and 72 hours.

Figure 6. Representative images of Live/Dead Assay for HOBs treated with 0, 1, 10 and 30 µg/ml of SDBS dispersed BNNTs after 24, 48 and 72 hours. Cytotoxicity was noted in cells treated with uncoated SDBS dispersed BNNTs at all concentrations and all time-points. Green-‐live, Red-‐dead. Scale bar = 50 µm.


Page 14 of 43

Page 15 of 43

Dead Cells

24 hours

48 hours

72 hours

Control 1 ug/ml 10 ug/ml 30 ug/ml

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


Figure 7. Representative images of Live/Dead Assay for HOBs treated with 0, 1, 10 and 30 µg/ml of PDBNNTs after 24, 48 and 72 hours. Relative to cells treated with non-functionalized BNNTs the observable number of non-viable cells was not significant at all concentrations. Green-live, Red-dead. Scale bar = 50 µm.

Quantification of cell viability was also performed as a function of fluorescence absorbance. Live/dead assay indicated that after 24, 48 and 72 hours of cellular exposure to increasing concentrations (1, 10 and 30 µg/ml) of non-functionalized BNNTs cell viability was significantly reduced relative to untreated control situations. However, the cell viability was not affected in HOBs cultured with PD-functionalized BNNT relative to control conditions. Indeed, a viability of approx. 90 % was maintained in all experimental groups after 72 hours. (Fig 8A). Similarly, cells exposed to non-functionalized and PDfunctionalized BNNT were observed to undergo differential metabolic activity and



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

proliferation rates. Critically, non-functionalized BNNTs significantly reduced both cellular metabolic activity and cell proliferation relative to control conditions. This effect was reversed in cells cultured with PD-functionalized BNNT (Fig 8B & C).

Figure 8. The influence of non-functionalized and PD-functionalized BNNTs on cellular viability, metabolic activity and proliferation. HOBs were observed following 24, 48 and 72 hours of culture with 1, 10 and 30 µg/ml of BNNTs. Cell viability as assed by Live/dead assay was significantly reduced in HOBs cultured with non-functionalized BNNT yet was unchanged in HOBs cultured with PD-functionalized BNNTs at all time points (A). Metabolic activity as assessed by alamarBlue® analysis indicated a trend of reduced metabolic activity in cells cultured with non-functionalized BNNTs relative to cells cultured with PD-functionalized BNNTs and under control culture conditions (B). Similarly, cellular proliferation as assessed via Pico Green assay indicated significant reduced cell proliferation in cells cultured in the presence of non-functionalized BNNTs relative to cells cultured with PD-functionalized BNNTs and under control culture conditions at concentrations of 30 µg/ml (C). Results were normalized to control samples for each time point. Results are expressed as mean ± SEM (n=5), * P

Effects of Polydopamine Functionalization on Boron Nitride Nanotube

Recommend Documents