Polymer Nanorod Hybrids: Potential

Subscriber access provided by UNIV OF LOUISIANA

Applications of Polymer, Composite, and Coating Materials

Liquid-Crystalline Hydroxyapatite/Polymer Nanorod Hybrids: Potential Bioplatform for Photodynamic Therapy and Cellular Scaffolds Masanari Nakayama, Wei Qi Lim, Satoshi Kajiyama, Akihito Kumamoto, Yuichi Ikuhara, Takashi Kato, and Yanli Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b02485 • Publication Date (Web): 23 Apr 2019 Downloaded from http://pubs.acs.org on April 28, 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 27 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 Materials & Interfaces

Liquid-Crystalline Hydroxyapatite/Polymer Nanorod Hybrids: Potential Bioplatform for Photodynamic Therapy and Cellular Scaffolds Masanari Nakayama,†,^ Wei Qi Lim,‡,^ Satoshi Kajiyama,† Akihito Kumamoto,§ Yuichi Ikuhara,§ Takashi Kato*,† and Yanli Zhao*,‡ †Department

of Chemistry and Biotechnology, School of Engineering, The University of Tokyo,

7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. ‡Division

of Chemistry and Biological Chemistry, School of Physical and Mathematical

Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371, Singapore. §Institute

of Engineering Innovation, School of Engineering, The University of Tokyo, 2-11-16

Yayoi, Bunkyo-ku, Tokyo 113-8656, Japan.

ABSTRACT: Recently, we found that self-organization of hydroxyapatite (HAp) with poly(acrylic acid) (PAA) leads to the formation of liquid-crystalline (LC) nanorod hybrids that form aligned films and show stimuli-responsive properties. Here we demonstrate that these biocompatible HAp/PAA hybrid nanorods represent a platform technology as drug nanocarriers for photodynamic cancer therapy and as bioscaffolds for the control of cellular alignment and

ACS Paragon Plus Environment

1

ACS Applied Materials & Interfaces 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 27

growth. To use hybrid nanorods as a drug nanocarrier, we introduced methylene blue (MB), a typical photosensitizer for photodynamic therapy, into the PAA nanolayer covering the surface of the HAp nanocrystals through electrostatic interactions. The stable MB-loaded HAp/PAA hybrid nanorods efficiently produced singlet oxygen from MB upon light irradiation and showed remarkable photodynamic therapeutic effects in cancer cells. Moreover, taking advantage of the mechanically responsive LC alignment properties of the HAp/PAA hybrid nanorods, macroscopically oriented bioscaffolds were prepared through a spin-coating process. The cells cultured on the oriented scaffolds showed cellular alignment and elongation along the oriented direction of the hybrid nanorods. The HAp/PAA hybrid nanorods demonstrate potential in drug delivery and tissue engineering. These unique LC HAp/PAA hybrid nanorods have significant potential as a platform for the development of various types of biomaterial.

KEYWORDS: photodynamic therapy; drug nanocarrier; cell culture scaffold; liquid crystal; biomineralization

In biominerals, such as seashells and bones, the morphologies and sizes of inorganic nanocrystals are precisely controlled through self-organization with biomacromolecules.1-3 Recently, we have developed liquid crystals based on CaCO3 and hydroxyapatite (HAp) nanorods through precise morphological control using an approach inspired by the formation of biominerals.4,5 These liquidcrystalline (LC) hybrid nanorods show stimuli-responsive alignment properties. HAp has attracted much attention as a biomedical material because of its high biocompatibility, bioactivity and bioresorbability.6 HAp-based materials have a variety of biomedical applications including use as


2

Page 3 of 27 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


nanocarriers for drug delivery and bioimaging,7-9 cell culture scaffolds10,11 and artificial bones.12 Likewise, LC HAp/poly(acrylic acid) (PAA) hybrid nanorods have potential as a platform technology for various types of biomaterial, because of their unique properties such as high colloidal stability, molecular-scale self-organized inorganic/polymer hybrid structures, stimuliresponsiveness, and facile synthetic procedures. Here we demonstrate a potential application of these new biocompatible LC HAp/PAA hybrid nanorods as a drug nanocarrier for photodynamic therapy (PDT) through incorporation of methylene blue (MB) into a PAA nanolayer on the surface of the hybrid nanorods. We also describe the advantages of the LC properties of the HAp/PAA hybrid nanorods as bioscaffolds with macroscopic orientation for cell culture (Figure 1). In the design of drug nanocarriers for PDT,13,14 the biocompatibility and colloidal stability of the HAp/PAA hybrid nanorods are useful properties in delivering drugs to tumors. In addition, the PAA nanolayer at the surface of the hybrid nanorods serves as a host layer for the incorporation of drug molecules, and especially positively charged molecules via electrostatic attraction. It is expected that PAA can interact with MB, which is a model drug molecule for a positively charged photosensitizer. HAp/PAA nanorods with incorporated MB are a good candidate for high performance PDT materials15-19 because MB permits the generation of singlet oxygen (1O2) upon light irradiation, which leads to the apoptosis or necrosis of cancer cells through oxidative stress.20,21 HAp/PAA nanorods also have potential as cell culture scaffolds to control cell growth, because of their LC ordering properties. Scaffolds composed of an oriented assembly of nanomaterials with anisotropic surface nanopatterning have been intensively studied for the alignment and


3


Page 4 of 27

morphological control of cells, which enhances cell differentiation and gene expression.22-27 Liquid crystals are promising candidates as oriented scaffold materials for the control of cell growth because various macroscopically ordered structures can be prepared through LC states.28-31 The LC HAp/PAA hybrid nanorods, combining the biocompatibility of HAp and alignment properties, are well-suited as oriented cell culture scaffolds.

Figure 1. Schematic illustration of HAp/PAA hybrid nanorods as a platform technology for the development of drug nanocarriers for PDT and bioscaffolds for cell growth control.


4

Page 5 of 27 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 The HAp/PAA hybrid nanorods (Figure 2a) were readily synthesized through the biomineralization-inspired crystallization of HAp in the presence of PAA.13 High-resolution transmission electron microscopy (HRTEM) image of the HAp/PAA hybrid nanorods revealed that the surface of HAp nanocrystal was functionalized with PAA (Figure 2b). In the electron energy-loss spectroscopy (EELS) analyses for the HAp/PAA hybrid nanorods, C-K core loss peaks were observed (Figure 2c), demonstrating the existence of PAA on the surface of HAp nanocrystals. Our aim is to functionalize the HAp/PAA hybrid nanorod as a drug nanocarrier through incorporation of drug molecules into the surface polymer nanolayer. MB as a photosensitizer was loaded onto HAp/PAA hybrid nanorods through a facile procedure in an aqueous solution. No significant difference in HAp structures was observed for nanorods synthesized in the presence and absence of MB. The average length of the MB-loaded HAp/PAA hybrid nanorods was 95 ± 30 nm and the average width was 22 ± 8 nm (Figure 3a and Figure S1). The characteristic nanorod X-ray diffraction (XRD) pattern and Fourier-transformed infrared (FTIR) spectrum confirmed the presence of HAp crystals (Figures S2 and S3). The peaks in the range 1400–1600 cm−1 in the FTIR spectrum are attributed to the carboxylate groups of PAA, supporting the existence of PAA on HAp surface. The MB-loaded nanorods formed stable colloidal states in aqueous solution (Figure 3a, inset). A zeta potential measurement showed that the surface of the hybrid nanorods had a negative potential of −29 ± 16 mV (Figure S4) after the incorporation of positively charged MB. The zeta potential for the MB-loaded HAp/PAA hybrid nanorods increased by 10 mV from the value of −39 ± 7 mV for the HAp/PAA hybrid nanorods


5


Page 6 of 27

without MB,4 suggesting the incorporation of MB into surface PAA nanolayer of the nanorods. Thermogravimetric (TG) measurements showed that the nanorod hybrids contained 15.1 wt% of the PAA and MB organic components (Figure S5). The weight ratio of the organic components to HAp increased by approximately 3 wt%, compared with that of HAp/PAA hybrid nanorods synthesized in the absence of MB.4 It is considered that this value corresponds to the amount of MB in the HAp/PAA nanorods. To quantify the amount of MB in the nanorod hybrids, MB-loaded HAp/PAA nanorod hybrids were dissolved in HCl aqueous solution, and the absorbance was measured (Figure S6). Based on a calibration curve, the loaded amount of MB in the hybrid nanorods was estimated to be 3.6 wt%, which is in agreement with


6

Page 7 of 27 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 2. Morphology of HAp/PAA hybrid nanorods. (a) Transmission electron microscopy (TEM) image of HAp/PAA hybrid nanorods. Inset is a photograph of an aqueous colloidal dispersion of HAp/PAA hybrid nanorods. (b) HRTEM image of HAp/PAA hybrid nanorods. (c) EELS spectrum of HAp/PAA hybrid nanorods. Inset shows the high-resolution EELS spectrum at the carbon K-edges for HAp/PAA hybrid nanorods and microgrid for comparison.


7


Page 8 of 27

Figure 3. Morphology and photophysical properties of MB-loaded HAp/PAA hybrid nanorods. (a) TEM image of MB-loaded HAp/PAA hybrid nanorods. Inset is a photograph of an aqueous colloidal dispersion of MB-loaded HAp/PAA hybrid nanorods. (b) Absorption spectra for a free MB solution and colloidal dispersions of MB-loaded HAp/PAA hybrid nanorods. (c) Photostability test for free MB and MB-loaded HAp/PAA hybrid nanorods. (d) 1O 2

generation, as assessed by measuring a change in DPBF absorption at 415 nm in the

presence of MB-loaded HAp/PAA hybrid nanorods and free MB, and in the absence of MB under light irradiation at 630 nm (100 mW/cm2).


8

Page 9 of 27 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


the TG result. Absorption and fluorescence spectra provide information about the MB state in the hybrid nanorods. The hybrid nanorods showed peaks at 570 nm, which was blue shifted from the absorbance at 665 nm for free MB (Figure 3b). A blue shift for the fluorescence peak was also observed (Figure S7). These results suggested that MB molecules were highly condensed in the nanorod hybrids to form H-aggregation.32,33 The PAA nanolayer on the surface of the hybrid nanorods serves as a host layer for condensed MB molecules through electrostatic interactions. To demonstrate the advantages of the MB-loaded HAp/PAA nanorod hybrids as a PDT drug nanocarrier, we conducted a photostability test. Photobleaching was suppressed for the hybrid nanorods compared with free MB (Figure 3c). Enzymatic reduction resistance tests were also performed (Figure S8), considering that MB can be easily reduced in biological environments to form colorless leucomethylene blue,34 which has no ability to produce 1O2. β-nicotinamideadenine dinucleotide, reduced dipotassium salt (NADH) and diaphorase were used for the reduction experiments35,36 and the reduction of MB was monitored by measuring MB absorbance. After 5 min, the reduction rate of MB was approximately 25% for the hybrid nanorods while that of free MB molecules was approximately 45%, suggesting that MB molecules in the MB-loaded HAp/PAA hybrid nanorods were protected from enzymatic reduction. These results show the potential of the hybrid nanorods for use as a nanocarrier for MB delivery. The capability of the hybrid nanorods to generate 1O2 was investigated using 1,3-diphenylisobenzofuran (DPBF), which is a typical probe molecule for the detection of 1O2 (Figure 3d). A decrease in the absorption peak at 415 nm because of the oxidization of DPBF by 1O2 was observed (Figure S9). In the presence of MB-loaded HAp/PAA hybrid nanorods, the 415 nm peak of DPBF decreased upon light irradiation at a wavelength of 630 nm, indicating the production of 1O2 from the hybrid nanorods. In comparison, the free MB generated higher amounts of 1O2, probably because O2 molecules were


9


Page 10 of 27

more accessible to the free MB molecules than to the MB molecules stacked in the MB-loaded HAp/PAA hybrid nanorods.37 However, we found that the hybrid nanorods exhibited pHresponsive MB release properties, leading to higher 1O2 production at low pH (Figure S10). Since the carboxylate groups of the PAA molecules are protonated in acidic conditions, MB molecules included in the hybrid nanorods through ionic interaction with PAA were released, resulting in an increase in the free MB concentration. The pH-responsive enhancement of 1O2 production in the hybrid nanorods is beneficial, as the intrinsic acidic environment in cancer cells would improve nanorod-mediated PDT.38-40 In addition, the nanocarrier showed remarkable colloidal stability. Even in cell culture media containing a high concentration of inorganic salts, stable colloidal states were formed (Figure S11a). The leakage of MB molecules from the hybrid nanorods was not significant even after incubation in cell culture medium for 6 h (Figure S11b). Their high colloidal stability enabled the hybrid nanorods to form condensed LC states without aggregation (Figure S12). Their self-assembly properties may broaden the application of the hybrid nanorods as a PDT agent carrier. Such paste-like LC materials contain high concentrations of PDT drugs and can be applied or injected to local sites for anti-bacterial coatings, and for the treatment of dental caries and cancerous skin, allowing for highly efficient and localized PDT. Encouraged by this promising PDT efficacy and stability, we went on to study the in-vitro therapeutic efficacy of the MB-loaded HAp/PAA hybrid nanorods. First, we investigated the cellular accumulation ability of the nanorods. The time-dependent cellular uptake of the MBloaded HAp/PAA hybrid nanorods in human cervical carcinoma cells (HeLa cells) was observed using confocal laser scanning microscopy (CLSM) (Figure 4). The cell nuclei were stained using 4’,6-diamidino-2-phenylindole (DAPI) before observation. Red fluorescence arising from MB molecules increased in HeLa cells with increasing MB-loaded HAp/PAA hybrid nanorod


10

Page 11 of 27 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


incubation time. These observations suggested that the hybrid nanorods could be internalized into the cells over time and subsequently used for PDT. Comparatively, negligible fluorescence can be observed from the confocal images of cells incubated with free MB for different time periods (Figure S13). This strongly implied the need of a delivery vehicle for the effective accumulation of molecular photosensitizers, underscoring the capability of the MB-loaded HAp/PAA hybrid nanorods for PDT.


11


Page 12 of 27

Figure 4. Time-dependent cellular uptake of MB-loaded HAp/PAA hybrid nanorods. CLSM images of cells treated with MB-loaded HAp/PAA hybrid nanorods (68 μg/mL) for 30 min (top row), 1 h (middle row) and 4 h (bottom row). MB shows red fluorescence and cell nuclei were stained using DAPI to show blue fluorescence.


12

Page 13 of 27 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 5. Intracellular 1O2 detection. CLSM images for cells (a) incubated with 17 μg/mL MB-loaded HAp/PAA hybrid nanorods for 4 h and then irradiated using light (100 mW/cm2) for 10 min, (b) treated using light irradiation (100 mW/cm2) for 10 min, (c) incubated with 17 μg/mL MB-loaded HAp/PAA hybrid nanorods for 4 h without light irradiation and (d) without any treatment. The cell nuclei were stained using Hoechst 33342 to show blue fluorescence. H2DCFDA was oxidized by 1O2 to show green fluorescence.

Figure 6. Cell viability of HeLa cells incubated for 24 h with different concentrations of MBloaded HAp/PAA hybrid nanorods with or without light irradiation.

The ability of the MB-loaded HAp/PAA hybrid nanorods to generate 1O2 in-vitro was next investigated (Figure 5). The cell nuclei were stained using Hoechst 33342 and the generation of 1O

2

was visualized using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). This probe emits

a green fluorescence after reaction with 1O2. For the cells treated using MB-loaded HAp/PAA hybrid nanorods and irradiated using light, strong green fluorescence signals were observed inside


13


Page 14 of 27

the cells (Figure 5a). However, in control experiments without MB-loaded HAp/PAA hybrid nanorods and/or light irradiation, negligible fluorescence was observed (Figure 5b–d). These results indicate that MB-loaded HAp/PAA hybrid nanorods can generate 1O2 inside cells upon light irradiation. Finally, cell photocytotoxicity was investigated using a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay (Figure 6). HeLa cells were treated using various concentrations of MB-loaded HAp/PAA hybrid nanorods. In dark conditions, no cytotoxicity was observed in the concentration range 0–68 μg/mL, indicating that the hybrid nanorods were highly biocompatible. In contrast, cell viability was significantly decreased on light irradiation with increasing concentrations of the hybrid nanorods. Notably, when the concentration of the hybrid nanorods was increased to 68 μg/mL, the cell viability decreased to approximately 5% after light irradiation. The photocytotoxicity of the MB-loaded HAp/PAA hybrid nanorods was comparable to that of free MB (Figure S14). The MB-loaded HAp/PAA hybrid nanorods produced efficient cancer cell cytotoxicity, demonstrating their potential as a nanocarrier for PDT.


14

Page 15 of 27 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. Preparation of macroscopically oriented bioscaffolds based on LC HAp/PAA hybrid nanorods. (a) Typical polarizing optical microscopy (POM) image of LC concentrated aqueous colloidal dispersions of HAp/PAA hybrid nanorods. (b) Schematic illustration of the preparation of HAp oriented bioscaffolds using LC HAp/PAA hybrid nanorods. (c) SEM image of oriented bioscaffolds for the region denoted by I in (d). (d) POM image of oriented bioscaffolds. (e) SEM image of oriented bioscaffolds for the region denoted by II in (d).


15


Page 16 of 27

Figure 8. Cell culture on the macroscopically oriented bioscaffolds. CLSM images of cells cultured on (a) HAp oriented bioscaffolds and (b) bare glass. Actin fibers were stained using RP to show orange fluorescence. Cell nuclei were stained using DAPI to show blue fluorescence. (c) Orientational order parameters of cells cultured on bare glass and HAp oriented scaffolds. (d, e) Polar plots of aspect ratios and orientation angles of cells cultured on (d) bare glass and (e) HAp oriented bioscaffolds.


16

Page 17 of 27 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


We went on to explore other potential biomedical applications for the HAp/PAA hybrid nanorods. We focused on the ability of the hybrid nanorods to form LC ordering because oriented assemblies of biocompatible nano-objects can be engineered to function as templates to control cell growth,22-27 leading to an enhancement in cell differentiation and function for tissue engineering. In general, the orientation control of HAp nanocrystals over macroscopic scale is challenging. Our approach involves exploiting the self-assembly of liquid crystals to achieve oriented HAp-based bioscaffolds. The HAp/PAA hybrid nanorods showed LC ordering in the concentrated aqueous colloidal dispersions (Figure 7a). We found that the LC nanorod hybrids can be macroscopically oriented through simple spin-coating processes and the oriented structures can be fixed on a glass substrate using poly(vinyl alcohol) (PVA) as a binder (Figure 7b). HAp/PAA hybrid nanorods spin-coated on a glass substrate covered with PVA remained on the glass substrate even after being washed with water or soaked in cell culture medium. When sufficient mechanical force was applied to the LC HAp/PAA hybrid nanorods by increasing the spinning rate of the spin-coating to 8000 rpm, a crossed birefringence pattern was observed over a centimeter scale under crossed polarizers (Figure 7d and Figure S15), indicating the macroscopic radial ordering of the nanorods. The thickness of the PVA layer and the HAp/PAA hybrid nanorod layer was estimated to be 1200 nm and 300 nm, respectively, based on a cross-sectional scanning electron microscopy (SEM) image (Figure S16). The orientation of the nanorod hybrids was directly observed using SEM (Figure 7c and 7e). The oriented directions of the nanorod hybrids at the regions denoted by I and II in Figure 7d were perpendicular to each other. This supports the radial orientation of the scaffolds. Having successfully prepared the oriented scaffolds, we investigated their potential to be used as a cell culture template. HeLa cells were cultured on the macroscopically oriented scaffolds and a


17


Page 18 of 27

bare glass substrate was used as a control. The cell cytoplasm was stained using rhodamine phalloidine (RP) and the cell nuclei were stained using DAPI. CLSM observations clearly showed that the cells cultured on the HAp/PAA hybrid nanorod oriented scaffolds were aligned along the oriented direction of the hybrid nanorods (Figure 8a). In contrast, no alignment was observed for the cells cultured on bare glass (Figure 8b). The orientational order parameter of the cells (S) was estimated using the following equation.

S=

< 3cos2 𝜃 ― 1 > 2

where θ is the orientation angle of the cells. For cells cultured on the oriented bioscaffolds, the orientational order parameter was calculated to be 0.80, which was twice that of cells cultured on bare glass (Figure 8c). The aspect ratio of cells cultured on the oriented scaffolds was 3.4 ± 1.8, which is higher than the aspect ratio of 2.5 ± 1.3 for cells cultured on a glass substrate (Figure S16). Figure 8d and 8e show the polar plots as a function of cell aspect ratio and growth angle for cells cultured on a bare glass substrate and oriented scaffolds, respectively. For the oriented scaffolds, cells were elongated and aligned along the oriented direction of the HAp/PAA hybrid nanorods, while such cellular alignment and elongation were not significant on the bare glass substrate. The oriented HAp/PAA hybrid nanorods functioned as cell culture scaffolds to control cellular alignment and morphology.

Conclusion We explored the biological properties of LC HAp/PAA hybrid nanorods, which have unique material characteristics such as biocompatibility, colloidal stability, stimuli-responsive LC


18

Page 19 of 27 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


ordering, molecular-scale inorganic/organic hybrid structures and facile environmentally-friendly synthetic procedures. We have developed nanocarriers for PDT and a bioscaffold for cell culture, based on the nanorod hybrids. MB molecules, as a model drug for PDT, were incorporated into the HAp/PAA hybrid nanorods through interaction with the surface PAA nanolayer. The MBloaded HAp/PAA hybrid nanorods produced 1O2 through light irradiation and showed an efficient photodynamic therapeutic effect in cancer cells. In addition, by taking advantage of the LC properties of the HAp/PAA hybrid nanorods, macroscopically oriented bioscaffolds were obtained. The oriented scaffolds induced cellular alignment and elongation along the oriented direction of the nanorod hybrids. The potential of the hybrid nanorods for drug delivery and tissue engineering was demonstrated. The smart LC HAp/PAA hybrid nanorods represent a novel nanoplatform for various biomedical applications.

ASSOCIATED CONTENT Supporting information is available free of charge on the ACS Publications website. (1) Methods. (2) Size distribution of MB-loaded HAp/PAA hybrid nanorods. (3) XRD pattern of MB-loaded HAp/PAA hybrid nanorods. (4) FTIR spectrum of MB-loaded HAp/PAA hybrid nanorods. (5) Zeta potential curve of MB-loaded HAp/PAA hybrid nanorods. (6) TG curve of MB-loaded HAp/PAA hybrid nanorods. (7) MB loading amount of MB-loaded HAp/PAA hybrid nanorods. (8) Fluorescence spectrum of MB-loaded HAp/PAA hybrid nanorods. (9) Enzymatic reduction test for MB-loaded HAp/PAA hybrid nanorods. (10) 1O2 generation by MB-loaded HAp/PAA


19


Page 20 of 27

hybrid nanorods. (11) MB release from MB-loaded HAp/PAA hybrid nanorods in acidic conditions. (12) Stability of MB-loaded HAp/PAA hybrid nanorods in cell culture medium. (13) LC properties of MB-loaded HAp/PAA hybrid nanorods. (14) Cellular free MB uptake. (15) Influence of spinning rate on the preparation of oriented scaffolds. (16) Cross-sectional SEM image of oriented scaffolds. (17) Aspect ratios of cells. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. (T. Kato) *E-mail: [email protected]. (Y. Zhao) Author Contributions ^M.N. and W.Q.L. contributed equally. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This study was partly supported by JSPS KAKENHI Grant Numbers JP15H02179 and JP17J09259. M.N. is grateful for financial support from a Japan Society for the Promotion of Science (JSPS) Research Fellowship for Young Scientists and the JSPS Program for Leading Graduate Schools (MERIT). The authors are grateful to Dr. Satoshi Yamaguchi for performing the zeta potential measurements. TEM observations were conducted at the Advanced Characterization Nanotechnology Platform at the University of Tokyo, which is supported by the “Nanotechnology


20

Page 21 of 27 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


Platform” of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This work was also partially supported by the Singapore National Research Foundation Investigatorship (No. NRF-NRFI2018-03). We thank Conn Hastings, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

REFERENCES (1) Nudelman, F.; Sommerdijk, N. A. J. M. Biomineralization as an Inspiration for Materials Chemistry. Angew. Chem. Int. Ed. 2012, 51, 6582–6596. (2) Arakaki, A.; Shimizu, K.; Oda, M.; Sakamoto, T.; Nishimura, T.; Kato, T. BiomineralizationInspired Synthesis of Functional Organic/Inorganic Hybrid Materials: Organic Molecular Control of Self-Organization of Hybrids. Org. Biomol. Chem. 2015, 13, 974–989. (3) Meldrum, F. C.; Cölfen, H. Controlling Mineral Morphologies and Structures in Biological and Synthetic Systems. Chem. Rev. 2008, 108, 4332–4432. (4) Nakayama, M.; Kajiyama, S.; Kumamoto, A.; Nishimura, T.; Ikuhara, Y.; Yamato, M.; Kato, T. Stimuli-Responsive Hydroxyapatite Liquid Crystal with Macroscopically Controllable Ordering and Magneto-Optical Functions. Nat. Commun. 2018, 9, 568. (5) Nakayama, M.; Kajiyama, S.; Nishimura, T.; Kato, T. Liquid-Crystalline Calcium Carbonate: Biomimetic Synthesis and Alignment of Nanorod Calcite. Chem. Sci. 2015, 6, 6230–6234. (6) Gómez-Morales, J.; Iafisco, M.; Delgado-López, J. M.; Sarda, S.; Drouet, C. Progress on the Preparation of Nanocrystalline Apatites and Surface Characterization: Overview of Fundamental and Applied Aspects. Prog. Cryst. Growth Charact. Mater. 2013, 59, 1–46.


21


Page 22 of 27

(7) Epple, M.; Ganesan, K.; Heumann, R.; Klesing, J.; Kovtun, A.; Neumann, S.; Sokolova, V. Application of Calcium Phosphate Nanoparticles in Biomedicine. J. Mater. Chem. 2010, 20, 18– 23. (8) Uskoković, V.; Uskoković, D. P. Nanosized Hydroxyapatite and Other Calcium Phosphates: Chemistry of Formation and Application as Drug and Gene Delivery Agents. J. Biomed. Mater. Res. Part B Appl. Biomater. 2011, 96B, 152–191. (9) Tabaković, A.; Kester, M.; Adair, J. H. Calcium Phosphate-Based Composite Nanoparticles in Bioimaging and Therapeutic Delivery Applications. WIREs Nanomed. Nanobiotechnol. 2012, 4, 96–112. (10) Chen, F.; Zhu, Y. J. Large-Scale Automated Production of Highly Ordered Ultralong Hydroxyapatite Nanowires and Construction of Various Fire-Resistant Flexible Ordered Architectures. ACS Nano 2016, 10, 11483–11495. (11) Holthaus, M. G.; Stolle, J.; Treccani, L.; Rezwan, K. Orientation of Human Osteoblasts on Hydroxyapatite-Based Microchannels. Acta Biomater. 2012, 8, 394–403. (12) Dorozhkin, S. V; Epple, M. Biological and Medical Significance of Calcilum Phosphates. Angew. Chem. Int. Ed. 2002, 41, 3130–3146. (13) Felsher, D. W. Cancer Revoked: Oncogenes as Therapeutic Targets. Nat. Rev. Cancer 2003, 3, 375–380.


22

Page 23 of 27 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


(14) Bechet, D.; Couleaud, P.; Frochot, C.; Viriot, M. L.; Guillemin, F.; Barberi-Heyob, M. Nanoparticles as Vehicles for Delivery of Photodynamic Therapy Agents. Trends Biotechnol. 2008, 26, 612–621. (15) Sahu, A.; Choi, W. Il; Lee, J. H.; Tae, G. Graphene Oxide Mediated Delivery of Methylene Blue for Combined Photodynamic and Photothermal Therapy. Biomaterials 2013, 34, 6239–6248. (16) Tardivo, J. P.; Del Giglio, A.; De Oliveira, C. S.; Gabrielli, D. S.; Junqueira, H. C.; Tada, D. B.; Severino, D.; De Fátima Turchiello, R.; Baptista, M. S. Methylene Blue in Photodynamic Therapy: From Basic Mechanisms to Clinical Applications. Photodiagnosis Photodyn. Ther. 2005, 2, 175–191. (17) Yan, F.; Zhang Y.; Kim, K. S.; Yuan, H.-K.; Vo-Dinh, Cellular Uptake and Photodynamic Activity of Protein Nanocages Containing Methylene Blue Photosensitizing Drug. Photochem. Photobiol. 2010, 86, 662–666. (18) Schwiertz, J.; Wiehe, A.; Gräfe, S.; Gitter, B.; Epple, M. Calcium Phosphate Nanoparticles as Efficient Carriers for Photodynamic Therapy against Cells and Bacteria. Biomaterials 2009, 30, 3324–3331. (19) Fan, W.; Qi, Y.; Wang, R.; Xu, C.; Zhao, N.; Xu, F. J. Calcium Carbonate-Methylene Blue Nanohybrids for Photodynamic Therapy and Ultrasound Imaging. Sci. China Life Sci. 2018, 61, 483–491. (20) Matés, J. M.; Sánchez-Jiménez, F. M. Role of Reactive Oxygen Species in Apoptosis: Implications for Cancer Therapy. Int. J. Biochem. Cell Biol. 2000, 32, 157–170.


23


Page 24 of 27

(21) Shi, H.; Ma, X.; Zhao, Q.; Liu, B.; Qu, Q.; An, Z.; Zhao, Y.; Huang, W. Ultrasmall Phosphorescent Polymer Dots for Ratiometric Oxygen Sensing and Photodynamic Cancer Therapy. Adv. Funct. Mater. 2014, 24, 4823–4830. (22) Nakayama, K. H.; Hong, G.; Lee, J. C.; Patel, J.; Edwards, B.; Zaitseva, T. S.; Paukshto, M. V.; Dai, H.; Cooke, J. P.; Woo, Y. J.; Huang, N. F. Aligned-Braided Nanofibrillar Scaffold with Endothelial Cells Enhances Arteriogenesis. ACS Nano 2015, 9, 6900–6908. (23) Lim, S. H.; Liu, X. Y.; Song, H.; Yarema, K. J.; Mao, H. Q. The Effect of Nanofiber-Guided Cell Alignment on the Preferential Differentiation of Neural Stem Cells. Biomaterials 2010, 31, 9031–9039. (24) Minami, K.; Kasuya, Y.; Yamazaki, T.; Ji, Q.; Nakanishi, W.; Hill, J. P.; Sakai, H.; Ariga, K. Highly Ordered 1D Fullerene Crystals for Concurrent Control of Macroscopic Cellular Orientation and Differentiation toward Large-Scale Tissue Engineering. Adv. Mater. 2015, 27, 4020–4026. (25) Wu, X.; Wang, S. Biomimetic Calcium Carbonate Concentric Microgrooves with Tunable Widths for Promoting MC3T3-E1 Cell Functions. Adv. Healthc. Mater. 2013, 2, 326–333. (26) Chung, W. J.; Oh, J. W.; Kwak, K.; Lee, B. Y.; Meyer, J.; Wang, E.; Hexemer, A.; Lee, S. W. Biomimetic Self-Templating Supramolecular Structures. Nature 2011, 478, 364–368. (27) Jin, H. E.; Jang, J.; Chung, J.; Lee, H. J.; Wang, E.; Lee, S. W.; Chung, W. J. Biomimetic Self-Templated Hierarchical Structures of Collagen-Like Peptide Amphiphiles. Nano Lett. 2015, 15, 7138–7145.


24

Page 25 of 27 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


(28) Kato, T.; Uchida, J.; Ichikawa, T.; Sakamoto, T. Functional Liquid Crystals towards the Next Generation of Materials. Angew. Chem. Int. Ed. 2018, 57, 4355–4371. (29) Kato, T.; Yoshio, M.; Ichikawa, T.; Soberats, B.; Ohno, H.; Funahashi, M. Transport of Ions and Electrons in Nanostructured Liquid Crystals. Nat. Rev. Mater. 2017, 2, 17001. (30) Bukusoglu, E.; Pantoja, M. B.; Mushenheim, P. C.; Wang, X.; Abbott, N. L. Design of Responsive and Active (Soft) Materials Using Liquid Crystals. Annu. Rev. Chem. Biomol. Eng. 2016, 7, 163–196. (31) Woltman, S. J.; Jay, G. D.; Crawford, G. P. Liquid-crystal materials find a new order in biomedical applications. Nat. Mater. 2007, 6, 929–938. (32) Bujdák, J.; Iyi, N.; Fujita, T. The Aggregation of Methylene Blue in Montmorillonite Dispersions. Clay Miner. 2002, 37, 121–133. (33) Bujdák, J.; Iyi, N.; Kaneko, Y.; Sasai, R. Molecular Orientation of Methylene Blue Cations Adsorbed on Clay Surfaces. Clay Miner. 2003, 38, 561–572. (34) May, J. M.; Qu, Z.; Cobb, C. E. Reduction and Uptake of Methylene Blue by Human Erythrocytes. Am. J. Physiol. Cell Physiol. 2004, 286, C1390–C1398. (35) Tang, W.; Xu, H.; Park, E. J.; Philbert, M. A.; Kopelman, R. Encapsulation of Methylene Blue in Polyacrylamide Nanoparticle Platforms Protects Its Photodynamic Effectiveness. Biochem. Biophys. Res. Commun. 2008, 369, 579–583.


25


Page 26 of 27

(36) He, X.; Wu, X.; Wang, K.; Shi, B.; Hai, L. Methylene Blue-Encapsulated PhosphonateTerminated Silica Nanoparticles for Simultaneous in Vivo Imaging and Photodynamic Therapy. Biomaterials 2009, 30, 5601–5609. (37) Jing, C.; Wang, R.; Ou, H.; Li, A.; An, Y.; Guo, S.; Shi, L. Axial Modification Inhibited HAggregation of Phthalocyanines in Polymeric Micelles for Enhanced PDT Efficacy. Chem. Commun. 2018, 54, 3985–3988. (38) Engin, K.; Leeper, D. B.; Cater, J. R.; Thistlethwaite, A. J.; Tupchong, L.; McFarlane, J. D. Extracellular PH Distribution in Human Tumours. Int. J. Hyperth. 1995, 11, 211–216. (39) Du, J. Z.; Sun, T. M.; Song, W. J.; Wu, J.; Wang, J. A Tumor-Acidity-Activated ChargeConversional Nanogel as an Intelligent Vehicle for Promoted Tumoral-Cell Uptake and Drug Delivery. Angew. Chem. Int. Ed. 2010, 49, 3621–3626. (40) Zhao, Y.; Luo, Z.; Li, M.; Qu, Q.; Ma, X.; Yu, S. H.; Zhao, Y. A Preloaded Amorphous Calcium Carbonate/Doxorubicin@silica Nanoreactor for pH-Responsive Delivery of an Anticancer Drug. Angew. Chem. Int. Ed. 2015, 54, 919–922. (41) Lentzen, M. Contrast Transfer and Resolution Limits for Sub-Angstrom High-Resolution Transmission Electron Microscopy. Microsc. Microanal. 2008, 14, 16–26.


26

Page 27 of 27 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


Table of Contents


27

Polymer Nanorod Hybrids: Potential

Recommend Documents