Multifunctional Photo- and Magnetoresponsive Graphene Oxide

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Biological and Medical Applications of Materials and Interfaces

Multifunctional Photo- and Magneto-responsive Graphene Oxide–Fe3O4 Nanocomposite–Alginate Hydrogel Platform for Ice Recrystallization Inhibition Yuan Cao, Muhammad Hassan, Yue Cheng, Zhongrong Chen, Meng Wang, Xiaozhang Zhang, Zeeshan Haider, and Gang Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b02887 • Publication Date (Web): 13 Mar 2019 Downloaded from http://pubs.acs.org on March 14, 2019

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ACS Applied Materials & Interfaces

Multifunctional Photo- and Magneto-responsive Graphene Oxide–Fe3O4 Nanocomposite– Alginate Hydrogel Platform for Ice Recrystallization Inhibition

Yuan Cao†, Muhammad Hassan§, Yue Cheng†, Zhongrong Chen†, Meng Wang†, Xiaozhang Zhang†, Zeeshan Haider†, Gang Zhao*†

†

Department of Electronic Science and Technology, University of Science and Technology of China, Hefei 230027, Anhui, China § Division of Nanomaterials & Chemistry, Hefei National Laboratory for Physical Sciences at the Microscale, Department of Chemistry, University of Science and Technology of China, Hefei 230026, China *Author

to whom correspondence should be addressed. Email: [email protected] (G.Z.) KEYWORDS: GO–Fe3O4 nanocomposites, alginate hydrogels, photo-thermal, magneto-thermal, ice recrystallization inhibition

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ABSTRACT: Tuning ice recrystallization (IR) has attracted tremendous interest in fundamental research and a variety of practical applications, including food and pharmaceutical engineering, fabrication of anti-icing coating and porous materials, and cryopreservation of biological cells and tissues. Although great efforts have been devoted for modulation of IR for better microstructure control of various materials, it still remains a challenge, especially in cryopreservation, where insufficient suppression of IR during warming is fatal to the cells. Herein, we report an all-in-one platform combing the external physical fields and the functional materials for both active and passive suppression of IR. Where, the photo- and magnetothermal dual-modal heating of GO– Fe3O4 nanocomposites (NCs) can be used to suppress IR with both enhanced global warming and microscale thermal disturbance. Moreover, the materials alginate hydrogels and GO–Fe3O4 NCs can act as IR inhibitors for further supression effect. As a typical application, we show that this GO–Fe3O4 nanocomposite–alginate hydrogel platform can succesfully enable low-cryoprotectant high-quality vitrification of stem cell-laden hydrogels. We believe that the versatile ice recrystallization inhibition (IRI) platform will have a profound influence on cryopreservation, and tremendously facilitate stem cell-based medicine to meet its ever-increasing demand in clinical.


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INTRODUCTON Ice recrystallization (IR), a ubiquitous phenomenon in nature, is of great interest and significant importance in the field of climate science1-2, pharmaceutical industry3, cryobiology4-5, and food science6. However, the mechanism of IR has not been fully understood and modulating the procedure of IR remains a challenge7. Nature has unique means of tuning ice formation, and inspired by the antifreeze proteins (AFPs) in polar fishes, great efforts have been devoted to investgating the mechanism of AFPs or synthetic AFPs mimics in ice recrystallization inhibitation (IRI)8. AFPs and some synthetic nano-/micro- particles were reported to controll ice formation by inhibiting ice growth and recrystallization (the growth of large crystals at the expense of smaller ices), which is especially important in biological cryopreservation7, 9. Actually, for vitrification cryopreservation, IR during thawing is a fatal factor to cell survival10-11. Nevertheless, the difficulty in the obtaining AFPs, and the unknown general standard for design as well as the potential hazard of AFP mimics restrict their clinical applications9. Recently, alginate hydrogel has emerged as an attractive candidate to suppress IR10, 12-14. Compared with the conventional IR inhibitors, due to the excellent biocompatibility, the alginate hydrogels as appealing three-dimensional scaffold materials can be directly delivered to human tissues15-20. Although the hydrogels can inhibit IR to a certain extent, the rewarming rate that has a pronounced effect on IR21 remains a limitation22. Recently, nanocomposite hydrogels find widespread applications due to their extraordinary properties that incorporate the novel performance of nanomaterials (photic, magnetic, and catalytic properties) into hydrogels23-27. The flexibility of embedded nanomaterials provides the nanocomposite hydrogels an innovative avenue to improve warming rate for tuning IR. Indeed, significant improvements in cryopreservation of bio-sample have been achieved by thermoresponsive magnetic Fe3O4 nanoparticles to suppress IR under alternating magnetic field (AMF)12, 28.

The application of AMF to magnetic nanoparticles is able to generate thermal via oscillation of 3 ACS Paragon Plus Environment


magnetic moment owing to Neel and Brownian relaxations29-32. Although promising, single magnetically-responsive thermal effect still restricts rewarming rate28, 33, which is insufficient to resist IR in ultra-low CPAs. Alternatively, near-infrared (NIR) light to trigger thermo-responsive is of particular interest, as it can be precisely and remotely tuned with light intensity34-38. NIR lightabsorbing nanostructures commonly involve carbon-based nanomaterials, gold nanoparticles, and copper semiconductor39. Of these, graphene oxide (GO) has attracted tremendous attention due to unique optical and electrical properties, excellent biocompatibility, and high conductivity40-43. It is not difficult to imagine that if dual stimuli-responses are combined, the reinforcement of heat rate and intensity could be obtained. Surprisingly, GO has been also demonstrated that it can mimic AFPs to greatly inhibit the ice crystals growth and recrystallization8, meanwhile the introduction of GO doesn’t lower the cell compatibility of the hydrogels35. We hypothesized that multiple IRI effects could be achieved by multifunctional nanocomposite hydrogels. Herein, we developed a GO–Fe3O4 nanocomposite–alginate hydrogel platform for controlling ice recrystallization by IR inhibitors and external physical fields. This approach enables lowcryoprotectant and high-quality vitrification of stem cell-laden nanocomposite hydrogels for further delivery. The alginate hydrogels and GO–Fe3O4 nanocomposites (NCs) can act as IR inhibitors; meanwhile, the NCs serve as localized heat sources to rapidly increase the hydrogel temperature actuated by NIR light and AMF for local inhibition of IR and rapid global rewarming. More interestingly, once the laser and magnetic field are turned off, heating induced by NCs immediately terminates to prevent overheating. In addition, the composite hydrogel showed good biocompatibility and the incorporation of highly ordered thermo-responsive nanomaterials can enhance the mechanical properties of the hydrogels44-45. Furthermore, the hydrogels can be assembled to produce 3D tissue constructs, and manipulated and accumulated at the target region through controlled magnetic guidance. Therefore, the novel approach can efficiently suppress IR 4 ACS Paragon Plus Environment

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and facilitate the applications of stem cell-based biocomposites for clinical therapy.



RESULTS AND DISCUSSION Ice Recrystallization of GO–Fe3O4 Nanocomposite–alginate Hydrogel Platform. Figure 1 shows a multifunctional platform for controlling ice recrystallization. In nature, some animals and plants have AFPs to protect them from freezing injury through regulating ice formation, including controlling ice nucleation, tuning ice crystals shape and growth, and suppressing IR7. Generally, several emerging strategies have been proposed for controlling IR. Among them, AFP mimics have attracted increasing research interests8. The AFPs and AFP mimics can preferentially bind to the basal and prism faces of ice crystals, causing local microcurvatures on the ice surface to inhibit ice growth7 (Figure 1a). Besides, the IR tune capacity of alginate hydrogels has stimulated substantial interest in cryopreservation. The alginate hydrogel microcapsules can effectively inhibit IR during warming10 (Figure 1b). Furthermore, an alternative novel strategy for suppressing IR is improving warming rate to decrease the formation of ice crystals during the rewarming. NIR light (Figure 1c) or AMF (Figure 1d) induced heating can be employed to assist warming, thereby suppressing IR12, 46.

In the study, we developed an all-in-one platform for controlling ice recrystallization by

functional materials and external physical fields (Figure 1e). The nano-in-micro platform based on photothermal and magnetothermal responses was illustrated in Figure S1 (Supporting Information). Two-dimensional GO sheets possess abundant oxygen-containing functional groups, including carboxyl, hydroxyl, carbonyl, and epoxy groups on its surface; these impart negative charge and can immobilize the Fe3+ and Fe2+ ions used in the preparation of Fe3O4 nanoparticles47. Therefore, Fe3O4 NPs were chemically deposited on the surface of GO nanosheets via the COOH-terminal, eventually generating GO–Fe3O4 NCs. The electrostatic spraying (ESS) offers high-throughput, low-cost, size-controllable particles over a narrow distribution; these maintained cell viability for mass manufacturing48-49. The GO–Fe3O4 nanocomposite–alginate hydrogel platform was fabricated by ESS, as shown in Figure S1a. Here, 6 ACS Paragon Plus Environment

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GO–Fe3O4 NCs, MSCs, and 2% (w/v) sodium alginate were used to generate monodisperse cellladen GO–Fe3O4 nanocomposite–alginate hydrogels (biocomposites) for further rewarming experiments (Movie S1, Supporting Information). The cooling process of biocomposites with plastic straw (PS) by vitrification, is well presented in Figure S1b and Movie S2. The biocomposites were collected and equilibrated in cryoprotectant (CPA) solutions. Afterward, the PS was plunged directly into liquid nitrogen for cooling. As illustrated in Figure S1c, the PS with biocomposites was rewarmed conventionally in a 37 °C water bath. Ice recrystallization inevitably appeared due to the low-pCPA and restrictive thermal conductivity (Movie S3). Alternatively, photic induction heating (PIH) and magnetic induction heating (MIH) were simultaneously manipulated for rapid and uniform rewarming of biocomposites (Figure S1d). The dual recovery platform includes 37 °C water in a 6-loops coil below an 808 nm NIR laser irradiation device. Characterization of GO–Fe3O4 NCs and Nanocomposite–alginate Hydrogels. The material properties of the GO–Fe3O4 NCs and nanocomposite–alginate hydrogels were systematically investigated (Figure 2). The morphology of the GO–Fe3O4 NCs was determined by TEM observations (Figure 2a). The abundant spherical Fe3O4 nanoparticles are decorated homogeneously on the surface of the two-dimensional GO nanosheets with no obvious aggregation47. As shown in Figure 2b, the size of GO–Fe3O4 NCs is ~614 nm. The morphology of the uniform nanocomposite hydrogels is shown in Figure 2c. Likewise, the size distribution of the nanocomposite hydrogels shows an average size of 319 µm with a range of 298-347 µm (Figure 2d). Low- and high-magnification SEM micrographs of as-prepared GO–Fe3O4 nanocomposite hydrogels with some wrinkles post low-CPA vitrification are shown in Figure 2e-f. The appearance of nanocomposite hydrogels post-vitrification is similar to pre-vitrification at low (Figure S2a) and high magnification(Figure S2b), suggesting that the integrity of the hydrogels can be maintained50. To analyze the atomic content of the hydrogels, SEM-EDS (dispersive X-ray 7 ACS Paragon Plus Environment


spectroscopy) analysis was performed without (Figure S3c) and with (Figure S3b) GO–Fe3O4 NCs. The results confirm the presence of iron and the increase of carbon and oxygen in the nanocomposite hydrogels. Fourier transform infrared (FT-IR) spectrum of GO, Fe3O4 and GO– Fe3O4 NCs are shown in Figure 2g. The spectra of GO reveals characteristic peaks are in good agreement with prior studies47, 51. The FT-IR spectrum of GO–Fe3O4, the characteristic peak at 1730 cm-1 corresponding to the stretching band of –COOH within the GO shifting to lower wavenumbers of 1564 cm-1 owing to the formation of -COO- after decorating with Fe3O4 NPs. Besides, the peak associated with the stretching of Fe-O bond in GO–Fe3O4 is shifted to lower 578 cm-1 compared with that of 597 cm-1 of Fe-O in pure Fe3O4, implying that Fe3O4 NPs are covalently bonded to the -COO- of GO nanosheets to form GO–Fe3O4 NCs52. Figure 2h shows the Raman spectra of GO and GO–Fe3O4 NCs. For GO, two strong peaks were located at around 1354 cm-1 and 1599 cm-1 that are corresponding to D and G band peaks, respectively. The D and G peaks of GO–Fe3O4 NCs occur at 1349 cm-1 and 1596 cm-1, which is shifted downward about 5 cm-1 and 3 cm-1 compared to those of GO. These phenomena are ascribed to the formation of Fe-O-C bonds between GO nanosheets and Fe3O4 NPs53. To study the magnetic properties of the as-prepared GO–Fe3O4 NCs, magnetic hysteresis curves were recorded, as displayed in Figure 2i. The saturation magnetization (Ms) is 48 emu·g-1 for GO–Fe3O4 and 70 emu·g-1 for bulk magnetic Fe3O4. The attraction and redispersion procedures of Fe3O4 and GO– Fe3O4 can be easily manipulated by an applied magnetic field (Figure S4) confirming the superparamagnetic behavior. More interestingly, the GO–Fe3O4 nanocomposite hydrogels also have magnetic properties48, as they can be assembled on a magnetic rod with round-shape (Figure S5). Therefore, the synthesized GO–Fe3O4 nanocomposite hydrogels are potential multifunctional materials for magnetically controlled drug delivery

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magnetic-controlled hydrogels patterning

assembly 55-56, , and photonic- and magnetic-induced biocomposite rewarming. To characterize the 8 ACS Paragon Plus Environment

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chemical composition of GO–Fe3O4 NCs, X-ray photoelectron spectroscopy (XPS) measurements were employed to detect the presence and position of C1s, O1s, and Fe2p at peaks of 285, 532, and 711 eV respectively. (Figure S6). The C1s peak in the XPS spectrum of GO–Fe3O4 NCs was deconvoluted into four peaks at 284.8, 286.3, 288.2, and 289.3 eV, respectively (Figure 2j)57. In the high-resolution Fe2p XPS spectrum of as-prepared GO–Fe3O4 NCs, as shown in Figure 2k, the binding energy peaks are at 710.6 and 724.6 eV corresponding to Fe2p3/2 and Fe2p1/2. The Fe2p3/2 peak can also be deconvoluted into separate components centered at 711.8 eV (Fe3+) and 710.3 eV (Fe2+). The relative areas of the resolved peak assigned to Fe3+ and Fe2+ were calculated to be 2.05 conforming the formation of Fe3O4 with Fe3+/Fe2+ ratio47. The mechanical properties of the GO–Fe3O4 nanocomposite–alginate hydrogels were investigated. The elastic modulus was calculated via the slope of the elastic zone of the tensile stress-strain curve (Figure 2l); it was approximately 1.4-fold higher than the hydrogels without the GO–Fe3O4 NCs, which is consistent with previous literatures58-59. Similarly, the compression testing also showed the mechanical property of hydrogels can be enhanced by incorporating nanomaterials (Figure S7). Therefore, the reinforcement of toughness of the hydrogels has advantages in delivery. Cell Viability and Proliferation Post-vitrification Based on IRI. To reduce cytotoxicity and osmotic injury, this study used a low concentration (2 M) of pCPAs. Ice recrystallization that is lethal for vitrified cells is common in low-CPA vitrification during warming60. Therefore, it is critical to design a strategy to achieve rapid recovery through reinforcing rewarming rate28. The ice inhibition effect of PIH and MIH on MSC in biocomposites, is well presented in Figure 3. To optimize the PIH and MIH conditions for biocomposites, the impact of the light intensity (0, 3, 4, and 5 W/cm2) and current intensity (0, 5, 15, and 25 A) on MSCs viability was fully investigated (Figure 3a). When the current intensity is 0 A, i.e., only PIH effect, the viability of microencapsulated MSCs increases as the light intensity varies from 3 to 5 W/cm2 post-vitrification, 9 ACS Paragon Plus Environment


demonstrating that individual PIH effect can improve the stem cell survival in the biocomposites. Similarly, the viability of the MSCs in the biocomposites also increases when the current intensity varies from 5 to 15 and 25 A post-vitrification without PIH. However, the viability for separate PIH or MIH is lower than 70%, which is acceptable criterion for therapy from FDA guidance61. This is probably because single PIH or MIH cannot generate sufficient heat to reduce IR for biocomposites during rewarming. Collectively, the combination of PIH and MIH is an appealing approach for local and global heating of vitrified biocomposites. The results show that the viability of MSCs in biocomposites is optimal at 4 W/cm2 and 15 A post-vitrification. Figure 3b shows the separate and combined effects of IR inhibitors, PIH, and MIH. The viability of the cell suspension is quite low (

Multifunctional Photo- and Magnetoresponsive Graphene Oxide

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