- Email: [email protected]
Sweet-MXene hydrogel with mixed-dimensional components for biomedical applications
Journal of the Mechanical Behavior of Biomedical Materials 101 (2020) 103440
Contents lists available at ScienceDirect
Journal of the Mechanical Behavior of Biomedical Materials journal homepage: www.elsevier.com/locate/jmbbm
Sweet-MXene hydrogel with mixed-dimensional components for biomedical applications
T
Rafieerad Alirezaa, Sequiera Glen Lestera, Yan Weianga, Kaur Parminderc, Amiri Ahmadb, Dhingra Sanjiva,∗ a Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, Regenerative Medicine Program, Department of Physiology and Pathophysiology, College of Medicine, University of Manitoba, Winnipeg, Canada b Department of Mechanical Engineering, Texas A&M University, College Station, TX, 77843, United States c University Institute of Engineering and Technology, Panjab University, Chandigarh, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Ti3C2 MXene nanosheets Porous hydrogel Electrically conductive hydrogel Stem cells Biocompatible composite
Biodegradable hydrogels are promising extracellular matrix-like materials for biomedical applications due to their high compatibility, ease of administration and minimal invasion. The injectable hydrogels to be considered for regenerative therapies should mimic the intrinsic properties of tissues, i.e. self-healing and swelling. Here, we present facile electrically conductive sweet-MXene (S-MXene) hydrogel with novel mixed-dimensional compositions including natural zero dimensional (0D) fluorescent carbon dots in honey, delaminated 2D fluorescent titanium carbide (Ti3C2) nanosheets and bioinspired 3D crosslinked polymeric chitosan networks. The developed versatile (Ti3C2-MXene-honey-chitosan) heterostructure exhibited excellent porous architecture with desired swelling and controlled degradation. This electrically conductive composite is highly biocompatible, it supported cell attachment and survival.
1. Introduction The soft hydrogel matrices with analogy to host tissues are becoming excellent choice for regenerative therapies by acting as bulking agent to cover the loss of host tissue after an injury or these can be used as a platform to deliver stem cells to support tissue regeneration. Recently, conductive biomaterials have emerged as promising choices to promote electrical conduction in nonconductive tissues in the body due to injury (Saravanan et al., 2018). After an injury, the post-treatment infection or immunological issues often compromise the life of patients or even lead to organ failure. Therefore, for successful clinical translation of biomaterials based approaches, there is a need to fabricate universal next generation multifunctional smart composites that not only promote regeneration but also address issues such as infection and inflammation. Therefore, the current study was aimed to fabricate a novel bioinspired, electrically conductive in nature, biocompatible, safe, antibacterial and anti-inflammatory hydrogel for tissue engineering. To obtain such ideal heterostructure, we fabricated a new mixeddimensional composite hydrogel by incorporating honey and titanium carbide MXene nanosheets- (Ti3C2) into a clinically approved polymer-
chitosan. To the best of our knowledge, this is the first study that combines honey and MXene nanosheets to synthesize a mixed-dimensional composite. We conceptualized the combination of these materials based on following three ideas. First, for long, honey has been traditionally used for its medicinal properties. It is an ancient remedy for nutrition deficiency and helps accelerate wound healing. The flavonoids and polyphenols, which are present in honey, perform as antiinflammatory, antibacterial and antioxidant agents. Besides its sugar contents (mono, di and oligo-saccharides), honey also contains bioactive compounds such as vitamins, amino acids and proteins which are important for cellular growth. Furthermore, anti-inflammatory potential of manuka honey (used in the synthesis of biocomposite in the current study) has been reported through the downregulation of proinflammatory cytokines e.g. TNF-α, MIP-1α, MIP-1β, IL-1β, IL-1α, IL-4, MMP-1, MMP-9 and FGF-13, and upregulation of anti-inflammatory cytokines e.g. MIP-3α, IL-10 and IL-8 (Minden-Birkenmaier et al., 2019; Tonks et al., 2003). Recently, the natural ubiquitous presence of fluorescent carbon nanodots (C-dots, average diameter: 3.2 ± 1.5 nm) in honey was reported (Mandani et al., 2017). These bioactive C-dots (zero dimensional or 0D) were found to be a fluorophore member in the carbon family with tunable emission surface properties.
∗ Corresponding author.Regenerative Medicine Program Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre R-3028-2, 351 Tache Avenue, Winnipeg, R2H2A6, Canada. E-mail address: [email protected] (S. Dhingra).
https://doi.org/10.1016/j.jmbbm.2019.103440 Received 25 May 2019; Received in revised form 21 August 2019; Accepted 17 September 2019 Available online 17 September 2019 1751-6161/ © 2019 Elsevier Ltd. All rights reserved.
Journal of the Mechanical Behavior of Biomedical Materials 101 (2020) 103440
A. Rafieerad, et al.
Fig. 1. (a, b) Scanning electron microscopic (SEM) images of delaminated Ti3C2 MXene nanosheets. (c) Transmission electron microscopic (TEM) image and (d) Energy dispersive X-ray spectroscopy (EDS) line scan of multilayer MXene nanosheets. The images depict proper exfoliation of uniform layers with approximately 200 nm wall-to-wall interlayer space and lattice d-spacing around 0.220 nm corresponding to MXene facet. EDS analysis highlights that main elements titanium and carbon were uniformly distributed within the nanosheets. (e-h) SEM based observations and EDS mapping of S-MXene hydrogel demonstrate highly porous 3D structure of S-MXene composite hydrogel with uniform distribution of pores. The pore size was in the range of 2.62–69.2 μm with well-defined elemental compositions (h). (i-l) TEM and high resolution TEM micrographs of composite. Selected area electron diffraction (SAED) and Fast Fourier Transform (FFT) patterns reveal the incorporation of mixed-dimensional components with different crystal lattices and d-spacing facets at a range of 0.200–0.330 nm (n = 3).
promote morphogenesis and tissue organization in a similar manner to that which occurs in natural tissue. Additionally, 0D carbon nanodots of honey in the composition of sweet-MXene hydrogel (as confirmed by our energy dispersive X-ray spectroscopic [EDS] data), will interact with the cells. Currently, there are no reports available on the synthesis of facile, honey and MXene based mixed-dimensional injectable and electrically conductive hydrogels for regenerative therapies. In this report, we present a mostly natural, biocompatible, mixed-dimensional, biodegradable, swellable and electrically conductive honey and MXene based (sweet-MXene or S-MXene) hydrogel with self-healing properties for multi-bioapplications. The detailed procedures for the synthesis, characterization and biological experiments are described in Supplementary Data. Briefly, the delamination of multilayered Ti3C2 MXene nanosheets from MAXphase Ti3AlC2 was chemically functionalized by hydrofluoric acid (HF) treatment. Ti3AlC2 powder was etched to significantly remove the aluminium (Al) species and form exfoliated Ti3C2 MXene nanosheets with fluorine and hydroxyl surface groups (Srimuk et al., 2016). The colloidal suspension of Ti3C2 MXene nanosheets and honey was incorporated into chitosan hydrogel to form a mixed 0, 2 and 3 dimensional composite. The scanning electron microscopic (SEM) images of MXene nanosheets depict proper delamination of uniform layers with approximately 200 nm wall-to-wall interlayer spacing (Fig. 1a and b). The transmission electron microscopic (TEM) micrographs revealed the architectural etching of MXene nanosheets morphology in uniform colloidal suspension (Fig. 1c). The crystal lattice of Ti3C2 MXene
Second, MXene is now considered as a booming carbon-based 2D material which is biocompatible, conductive in nature, and has antibacterial properties (Huang et al., 2018). We recently reported that Ti3C2 MXene based biomaterials possess intrinsic anti-inflammatory and immunomodulatory properties. In this study, we found that Ti3C2 quantum dots selectively suppress activation of CD4+IFN-γ+ T-lymphocytes and promote expansion of immunosuppressive CD4+CD25+FoxP3+ regulatory T. Furthermore, we reported that MXene quantum dots were able to downregulate the levels of inflammatory cytokines TNF-α and IFN-γ, also there was an increasing trend in the levels of anti-inflammatory cytokine IL-10 (Rafieerad et al., 2019; Soleymaniha et al., 2019). Moreover, Ti3C2 MXene nanosheets with enhanced surface properties have been found to be electrically conductive and possess autofluorescence properties (Wang et al., 2017). Third, the application of chitosan based 3D polysaccharide hydrogels and scaffolds in regenerative medicine is widely reported (Suh and Matthew, 2000). Chitosan is a biocompatible, biodegradable and clinically accepted polymer. Therefore, addition of electrically conductive, anti-bacterial and anti-inflammatory agents can provide a wide avenue to biomedical applications of chitosan based 3D biomaterials. Furthermore, fabrication of a mixed-dimensional hydrogel composite has several advantages. For example, two-dimensional (2D) planar and well-defined geometry of MXene nanosheets will provide large surface area for cell attachment and growth. Also, addition of 2D carbide based material in a composite enhances its surface energy, hydrophilicity and conductivity (Zhang et al., 2018). Whereas, the 3D architecture of chitosan will mimic native tissue in the body. Also, biomaterials based 3D assemblies facilitate cellular interactions that 2
Journal of the Mechanical Behavior of Biomedical Materials 101 (2020) 103440
A. Rafieerad, et al.
Fig. 2. Physico-mechanical properties of the synthesized S-MXene hydrogels containing low molecular weight (L) and medium molecular weight (M) chitosan. (a, b) Swelling ability and water absorption capacity of the hydrogels were measured by immersing the scaffold in DMEM for 1 h (yellow) and 24 h (brown). (c) Degradation rate of the composite hydrogels (S-MXene L-brown; S-MXene M-yellow) in lysozyme containing medium was measured for 96 h at 37 °C. (d) Electrical resistivity (yellow) and conductivity (brown) of aqueous Ti3C2 MXene, S-MXene L and S-MXene M hydrogels was assessed by measuring resistivity of the composites by digital multimeter. (e-n) Long-term stability of the crosslinked S-MXene L (e,g,i,j) and S-MXene M (f,h,k,l) hydrogels at 37 °C was determined by incubating the biocomposites with or without culture medium. The hydrogel architecture was intact for 2 weeks in the presence or absence of medium at 37 °C. (m,n) Self-healing potential was measured by cutting the S-MXene hydrogels (L and M respectively) into two pieces and incubating these pieces together at 37 °C for 30 min. At the end of incubation period the hydrogel was able to heal itself. (o) Injectability of S-MXene L and M hydrogels (left to right) was tested using a 26 g needle and 1 ml syringe. (n = 3). 3
Journal of the Mechanical Behavior of Biomedical Materials 101 (2020) 103440
A. Rafieerad, et al.
Fig. 3. Assessment of autofluorescence of the hydrogel. MXene nanosheets and carbon dots present in honey naturally display autofluorescence properties, which can be exploited for imaging, tracking, biosensors related applications. (a-d) The autofluorescence of Ti3C2 MXene nanosheets (a,b) and S-MXene hydrogel (c,d) was detected by Cytation 5 imaging system at different excitation/emission wavelengths; Dapi (377 nm, 447 nm), GFP (469 nm, 525 nm), Texas Red (586 nm, 647 nm) and Cy5 (628 nm, 685 nm) as well as the combined (merged) images of all four filters. The MXene nanosheets and C-dots incorporated in chitosan architecture displayed autofluorescence at different wavelengths.
nanostructures into chitosan polymers can presumably limit the mobility of chitosan chains depending on the surface adsorption of functional groups, also it reduces the crystalline phase with weak crystal rings (Chaudhry and Mittal, 2013; Justin et al., 2015). The physicochemical properties of the S-MXene composite including swelling ability, degradation rate, stability, electrical conductivity and self-healing nature were measured as described in supplementary methods. Generally, addition of carbon-based nanocomposites into the structure of polymers e.g. chitosan, potentially enhances the mechanical properties (Baradaran et al., 2014; Lipatov et al., 2018; Qiu and Wang, 2011; Young et al., 2018; Zhang et al., 2016). On the other hand, the molecular weight of chitosan hydrogel is also reported to have a direct effect on mechanical stability of the composite (Huei and Hwa, 1996). To this end, in the current study, we fabricated S-MXene hydrogel with low molecular weight chitosan (S-MXene L) as well as medium molecular weight S-MXene (S-MXene M). We compared the physico-mechanical behavior of both S-MXene L and S-MXene M hydrogels. There were no significant changes found in the swelling ability, biodegradation rate, stability, self-healing and injectability of these two biocomposites. Therefore, presence of MXene nanosheets preserves mechanical properties of the chitosan based composite. We found that S-MXene hydrogels displayed excellent swelling ability (Fig. 2a and b). It is biodegradable, self-healing and electrically conductive in nature (Fig. 2c–p). It is already reported that Ti3C2 MXene as well as honey (due to the presence of C-Dots) display autofluorescence properties, which can be exploited for imaging, tracking, biosensors related applications (Zhou et al., 2017). In the current study, the MXene nanosheets separately as well as S-MXene hydrogel displayed excellent autofluorescence at different excitation/emission wavelengths; Dapi (377 nm, 447 nm), GFP (469 nm, 525 nm), Texas Red (586 nm,
nanosheets confirmed d-spacing around 0.220 nm corresponding to MXene facets. The EDS analysis further confirmed the removal of Al from MAX phase with well-defined elemental compositions of multilayer Ti3C2 and –NH group (Fig. 1d; Supplementary Fig. S1). Our data also demonstrate a uniform distribution of carbon (C) and titanium (Ti) elements with negligible traces of Al (Fig. 1d; Supplementary Fig. S1). Additionally, the structure of Ti3C2 MXene material also includes notable amounts of nitrogen (N), fluorine (F) and oxygen (O) following exfoliation with HF (Halim et al., 2016). The SEM images of S-MXene hydrogel composite confirmed highly-porous crosslinked networks composite with the pore size in the range of 2.62 μm–69.2 μm (Fig. 1e and f). The porous network of a biomaterial is important to exhibit sufficient swelling kinetic and facilitate oxygen and nutrients transfer to the cells. The EDS analysis detected uniform distribution of different elements of honey and MXene in the hydrogel (Fig. 1g and h; Supplementary Fig. S2). The high-resolution TEM and selected area electron diffraction (SAED) micrographs confirmed clear multi-dimensional crystalline patterns of S-MXene hydrogel composite (Fig. 1i–k). The fast fourier transform (FFT) images revealed the amorphous phase transmission of crystal lattices at an approximate range of 0.200–0.330 nm (Fig. 1l; Supplementary Fig. S3). It is important to note that the crystal phase in the structure of S-MXene hydrogel was attributed to crystalline lattice of Ti3C2 MXene nanosheets. Our results indicate that, the lateral d-spacing corresponds to Ti3C2 MXene (1 0 5) and Ti atom (1 1 2 0) facets (Li et al., 2017; Luo et al., 2017; Mashtalir et al., 2013; Xu et al., 2018). Our TEM results of S-MXene hydrogel also show d-spacing lattice of around 2.0 Å and 3.3 Å for MXene and carbon dots respectively (Supplementary Fig. S3), which is in agreement with the reported range in literature. Furthermore, studies on the crystal analysis of chitosan based composites found that addition of carbon 4
Journal of the Mechanical Behavior of Biomedical Materials 101 (2020) 103440
A. Rafieerad, et al.
Fig. 4. Biocompatibility of the S-MXene hydrogel with (a) rat bone marrow derived mesenchymal stem cells (MSCs) and (b) human iPSCs derived cardiomyocytes was assessed by determining cell viability using a fluorescent dye Calcein AM. The green fluorescent Calcein AM will stain only live cells. Dapi was used to stain nucleus. (c) MSCs after 24 h and (d) cardiomyocytes after 48 h of culture in the presence of S-MXene hydrogel showed excellent viability. Therefore, the hydrogel is compatible with both cell types. Interestingly, we also found a statistically significant increase in the number of live cells in case of iPSC derived cardiomyocytes compared to control group. *p < 0.05 compared to control group. (n = 3–6). Data are expressed as mean ± SD.
cardiomyocytes and neurons are reported to worsen the disease pathophysiology during cardiovascular and neurological disorders. It is therefore conceivable that a mixed-dimensional- Ti3C2-MXene-honeychitosan biocomposite might offer additional benefits over a chitosan based hydrogel or scaffold when applied for tissue regeneration. Therefore, a multifunctional electrically conductive novel S-MXene hydrogel, which is compatible with different types of stem cells, and exert inherent antibacterial and anti-inflammatory properties, may have a great potential in facilitating tissue repair in several degenerative diseases such as cardiovascular, neurological and autoimmune diseases.
647 nm), Cy5 (628 nm, 685 nm) (Fig. 3). Next, we assessed the biocompatibility of S-MXene hydrogel. The composite displayed no cytotoxicity toward rat bone marrow derived mesenchymal stem cells (MSCs) and human induced pluripotent stem cells (iPSCs) derived cardiomyocytes (Fig. 4a–d). Bone marrow derived MSCs and iPSCs are already being tested in animal studies and Phase I and II clinical trials for cardiac regeneration, neurological disorders, diabetes, orthopedic diseases and autoimmune disorders (Trounson and McDonald, 2015). One of the challenges in clinical translation of stem cell therapy is poor survival of transplanted cells in the recipient. In the current study, we found that the S-MXene hydrogel was not only safe for the stem cells, also it promoted survival of iPSCs-derived cardiomyocytes (4b,d). In our ongoing studies we are testing the role of Ti3C2 MXene based materials on differentiation potential of iPSCs to cardiomyocytes. The elevated inflammation is a hall mark in case of degenerative diseases. Also, bacterial infections are reported to be associated with the pathogenesis of autoimmune diseases, cardiovascular disorders and other degenerative diseases (El Kholy et al., 2015; Kivity et al., 2009). Furthermore, conduction abnormalities in electrical signals among
Acknowledgment This work was supported by research grants from Canadian Institutes of Health Research.
5
Journal of the Mechanical Behavior of Biomedical Materials 101 (2020) 103440
A. Rafieerad, et al.
Appendix A. Supplementary data
Luo, J., Zhang, W., Yuan, H., Jin, C., Zhang, L., Huang, H., Liang, C., Xia, Y., Zhang, J., Gan, Y., 2017. Pillared structure design of MXene with ultralarge interlayer spacing for high-performance lithium-ion capacitors. ACS Nano 11, 2459–2469. Mandani, S., Dey, D., Sharma, B., Sarma, T.K., 2017. Natural occurrence of fluorescent carbon dots in honey. Carbon 119, 569–572. Mashtalir, O., Naguib, M., Mochalin, V.N., Dall'Agnese, Y., Heon, M., Barsoum, M.W., Gogotsi, Y., 2013. Intercalation and delamination of layered carbides and carbonitrides. Nat. Commun. 4 1716. Minden-Birkenmaier, B.A., Meadows, M.B., Cherukuri, K., Smeltzer, M.P., Smith, R.A., Radic, M.Z., Bowlin, G.L., 2019. The effect of manuka honey on dHL-60 cytokine, chemokine, and matrix-degrading enzyme release under inflammatory conditions. Med one 4. Qiu, J., Wang, S., 2011. Enhancing polymer performance through graphene sheets. J. Appl. Polym. Sci. 119, 3670–3674. Rafieerad, A., Yan, W., Sequiera, G.L., Sareen, N., Abu‐El‐Rub, E., Moudgil, M., Dhingra, S., 2019. Application of Ti3C2 MXene quantum dots for immunomodulation and regenerative medicine. Adv. Healthc. Mater 1900569. Saravanan, S., Sareen, N., Abu-El-Rub, E., Ashour, H., Sequiera, G.L., Ammar, H.I., Gopinath, V., Shamaa, A.A., Sayed, S.S.E., Moudgil, M., 2018. Graphene oxide-gold nanosheets containing chitosan scaffold improves ventricular contractility and function after implantation into infarcted heart. Sci. Rep. 8 15069. Soleymaniha, M., Shahbazi, M.A., Rafieerad, A.R., Maleki, A., Amiri, A., 2019. Promoting role of MXene nanosheets in biomedical sciences: therapeutic and biosensing innovations. Adv. Healthc. Mater. 8 1801137. Srimuk, P., Kaasik, F., Krüner, B., Tolosa, A., Fleischmann, S., Jäckel, N., Tekeli, M.C., Aslan, M., Suss, M.E., Presser, V., 2016. MXene as a novel intercalation-type pseudocapacitive cathode and anode for capacitive deionization. J. Mater. Chem. 4, 18265–18271. Suh, J.-K.F., Matthew, H.W., 2000. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. Biomaterials 21, 2589–2598. Tonks, A.J., Cooper, R., Jones, K., Blair, S., Parton, J., Tonks, A., 2003. Honey stimulates inflammatory cytokine production from monocytes. Cytokine 21, 242–247. Trounson, A., McDonald, C., 2015. Stem cell therapies in clinical trials: progress and challenges. Cell stem cell 17, 11–22. Wang, Z., Xuan, J., Zhao, Z., Li, Q., Geng, F., 2017. Versatile cutting method for producing fluorescent ultrasmall MXene sheets. ACS Nano 11, 11559–11565. Xu, Q., Ding, L., Wen, Y., Yang, W., Zhou, H., Chen, X., Street, J., Zhou, A., Ong, W.-J., Li, N., 2018. High photoluminescence quantum yield of 18.7% by using nitrogen-doped Ti 3 C 2 MXene quantum dots. J. Mater. Chem. C 6, 6360–6369. Young, R.J., Liu, M., Kinloch, I.A., Li, S., Zhao, X., Vallés, C., Papageorgiou, D.G., 2018. The mechanics of reinforcement of polymers by graphene nanoplatelets. Compos. Sci. Technol. 154, 110–116. Zhang, H., Wang, L., Chen, Q., Li, P., Zhou, A., Cao, X., Hu, Q., 2016. Preparation, mechanical and anti-friction performance of MXene/polymer composites. Mater. Des. 92, 682–689. Zhang, Y.-Z., Lee, K.H., Anjum, D.H., Sougrat, R., Jiang, Q., Kim, H., Alshareef, H.N., 2018. MXenes stretch hydrogel sensor performance to new limits. Science Advances 4, eaat0098. Zhou, L., Wu, F., Yu, J., Deng, Q., Zhang, F., Wang, G., 2017. Titanium carbide (Ti3C2Tx) MXene: a novel precursor to amphiphilic carbide-derived graphene quantum dots for fluorescent ink, light-emitting composite and bioimaging. Carbon 118, 50–57.
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jmbbm.2019.103440. Author contributions A.R, A.A, and S.D. conceptualized the study; A.R, GLS, P.K, A.A and SD designed the experiments; A.R, GLS, W.Y and P.K carried out experiments and acquired the data; A.R, GLS, P.K and SD interpreted the data, carried out data analysis and drafted the manuscript. Conflicts of interest Authors declare no competing interests. References Baradaran, S., Moghaddam, E., Basirun, W.J., Mehrali, M., Sookhakian, M., Hamdi, M., Moghaddam, M.N., Alias, Y., 2014. Mechanical properties and biomedical applications of a nanotube hydroxyapatite-reduced graphene oxide composite. Carbon 69, 32–45. Chaudhry, A., Mittal, V., 2013. High‐density polyethylene nanocomposites using masterbatches of chlorinated polyethylene/graphene oxide. Polym. Eng. Sci. 53, 78–88. El Kholy, K., Genco, R.J., Van Dyke, T.E., 2015. Oral infections and cardiovascular disease. Trends Endocrinol. Metab. 26, 315–321. Halim, J., Cook, K.M., Naguib, M., Eklund, P., Gogotsi, Y., Rosen, J., Barsoum, M.W., 2016. X-ray photoelectron spectroscopy of select multi-layered transition metal carbides (MXenes). Appl. Surf. Sci. 362, 406–417. Huang, K., Li, Z., Lin, J., Han, G., Huang, P., 2018. Two-dimensional Transition Metal Carbides and Nitrides (MXenes) for Biomedical Applications. Chemical Society Reviews. Huei, C.R., Hwa, H.-D., 1996. Effect of molecular weight of chitosan with the same degree of deacetylation on the thermal, mechanical, and permeability properties of the prepared membrane. Carbohydr. Polym. 29, 353–358. Justin, R., Román, S., Chen, D., Tao, K., Geng, X., Grant, R.T., MacNeil, S., Sun, K., Chen, B., 2015. Biodegradable and conductive chitosan–graphene quantum dot nanocomposite microneedles for delivery of both small and large molecular weight therapeutics. RSC Adv. 5, 51934–51946. Kivity, S., Agmon-Levin, N., Blank, M., Shoenfeld, Y., 2009. Infections and autoimmunity–friends or foes? Trends Immunol. 30, 409–414. Li, R., Zhang, L., Shi, L., Wang, P., 2017. MXene Ti3C2: an effective 2D light-to-heat conversion material. ACS Nano 11, 3752–3759. Lipatov, A., Lu, H., Alhabeb, M., Anasori, B., Gruverman, A., Gogotsi, Y., Sinitskii, A., 2018. Elastic properties of 2D Ti3C2Tx MXene monolayers and bilayers. Science Advances 4, eaat0491.
6
Contents lists available at ScienceDirect
Journal of the Mechanical Behavior of Biomedical Materials journal homepage: www.elsevier.com/locate/jmbbm
Sweet-MXene hydrogel with mixed-dimensional components for biomedical applications
T
Rafieerad Alirezaa, Sequiera Glen Lestera, Yan Weianga, Kaur Parminderc, Amiri Ahmadb, Dhingra Sanjiva,∗ a Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, Regenerative Medicine Program, Department of Physiology and Pathophysiology, College of Medicine, University of Manitoba, Winnipeg, Canada b Department of Mechanical Engineering, Texas A&M University, College Station, TX, 77843, United States c University Institute of Engineering and Technology, Panjab University, Chandigarh, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Ti3C2 MXene nanosheets Porous hydrogel Electrically conductive hydrogel Stem cells Biocompatible composite
Biodegradable hydrogels are promising extracellular matrix-like materials for biomedical applications due to their high compatibility, ease of administration and minimal invasion. The injectable hydrogels to be considered for regenerative therapies should mimic the intrinsic properties of tissues, i.e. self-healing and swelling. Here, we present facile electrically conductive sweet-MXene (S-MXene) hydrogel with novel mixed-dimensional compositions including natural zero dimensional (0D) fluorescent carbon dots in honey, delaminated 2D fluorescent titanium carbide (Ti3C2) nanosheets and bioinspired 3D crosslinked polymeric chitosan networks. The developed versatile (Ti3C2-MXene-honey-chitosan) heterostructure exhibited excellent porous architecture with desired swelling and controlled degradation. This electrically conductive composite is highly biocompatible, it supported cell attachment and survival.
1. Introduction The soft hydrogel matrices with analogy to host tissues are becoming excellent choice for regenerative therapies by acting as bulking agent to cover the loss of host tissue after an injury or these can be used as a platform to deliver stem cells to support tissue regeneration. Recently, conductive biomaterials have emerged as promising choices to promote electrical conduction in nonconductive tissues in the body due to injury (Saravanan et al., 2018). After an injury, the post-treatment infection or immunological issues often compromise the life of patients or even lead to organ failure. Therefore, for successful clinical translation of biomaterials based approaches, there is a need to fabricate universal next generation multifunctional smart composites that not only promote regeneration but also address issues such as infection and inflammation. Therefore, the current study was aimed to fabricate a novel bioinspired, electrically conductive in nature, biocompatible, safe, antibacterial and anti-inflammatory hydrogel for tissue engineering. To obtain such ideal heterostructure, we fabricated a new mixeddimensional composite hydrogel by incorporating honey and titanium carbide MXene nanosheets- (Ti3C2) into a clinically approved polymer-
chitosan. To the best of our knowledge, this is the first study that combines honey and MXene nanosheets to synthesize a mixed-dimensional composite. We conceptualized the combination of these materials based on following three ideas. First, for long, honey has been traditionally used for its medicinal properties. It is an ancient remedy for nutrition deficiency and helps accelerate wound healing. The flavonoids and polyphenols, which are present in honey, perform as antiinflammatory, antibacterial and antioxidant agents. Besides its sugar contents (mono, di and oligo-saccharides), honey also contains bioactive compounds such as vitamins, amino acids and proteins which are important for cellular growth. Furthermore, anti-inflammatory potential of manuka honey (used in the synthesis of biocomposite in the current study) has been reported through the downregulation of proinflammatory cytokines e.g. TNF-α, MIP-1α, MIP-1β, IL-1β, IL-1α, IL-4, MMP-1, MMP-9 and FGF-13, and upregulation of anti-inflammatory cytokines e.g. MIP-3α, IL-10 and IL-8 (Minden-Birkenmaier et al., 2019; Tonks et al., 2003). Recently, the natural ubiquitous presence of fluorescent carbon nanodots (C-dots, average diameter: 3.2 ± 1.5 nm) in honey was reported (Mandani et al., 2017). These bioactive C-dots (zero dimensional or 0D) were found to be a fluorophore member in the carbon family with tunable emission surface properties.
∗ Corresponding author.Regenerative Medicine Program Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre R-3028-2, 351 Tache Avenue, Winnipeg, R2H2A6, Canada. E-mail address: [email protected] (S. Dhingra).
https://doi.org/10.1016/j.jmbbm.2019.103440 Received 25 May 2019; Received in revised form 21 August 2019; Accepted 17 September 2019 Available online 17 September 2019 1751-6161/ © 2019 Elsevier Ltd. All rights reserved.
Journal of the Mechanical Behavior of Biomedical Materials 101 (2020) 103440
A. Rafieerad, et al.
Fig. 1. (a, b) Scanning electron microscopic (SEM) images of delaminated Ti3C2 MXene nanosheets. (c) Transmission electron microscopic (TEM) image and (d) Energy dispersive X-ray spectroscopy (EDS) line scan of multilayer MXene nanosheets. The images depict proper exfoliation of uniform layers with approximately 200 nm wall-to-wall interlayer space and lattice d-spacing around 0.220 nm corresponding to MXene facet. EDS analysis highlights that main elements titanium and carbon were uniformly distributed within the nanosheets. (e-h) SEM based observations and EDS mapping of S-MXene hydrogel demonstrate highly porous 3D structure of S-MXene composite hydrogel with uniform distribution of pores. The pore size was in the range of 2.62–69.2 μm with well-defined elemental compositions (h). (i-l) TEM and high resolution TEM micrographs of composite. Selected area electron diffraction (SAED) and Fast Fourier Transform (FFT) patterns reveal the incorporation of mixed-dimensional components with different crystal lattices and d-spacing facets at a range of 0.200–0.330 nm (n = 3).
promote morphogenesis and tissue organization in a similar manner to that which occurs in natural tissue. Additionally, 0D carbon nanodots of honey in the composition of sweet-MXene hydrogel (as confirmed by our energy dispersive X-ray spectroscopic [EDS] data), will interact with the cells. Currently, there are no reports available on the synthesis of facile, honey and MXene based mixed-dimensional injectable and electrically conductive hydrogels for regenerative therapies. In this report, we present a mostly natural, biocompatible, mixed-dimensional, biodegradable, swellable and electrically conductive honey and MXene based (sweet-MXene or S-MXene) hydrogel with self-healing properties for multi-bioapplications. The detailed procedures for the synthesis, characterization and biological experiments are described in Supplementary Data. Briefly, the delamination of multilayered Ti3C2 MXene nanosheets from MAXphase Ti3AlC2 was chemically functionalized by hydrofluoric acid (HF) treatment. Ti3AlC2 powder was etched to significantly remove the aluminium (Al) species and form exfoliated Ti3C2 MXene nanosheets with fluorine and hydroxyl surface groups (Srimuk et al., 2016). The colloidal suspension of Ti3C2 MXene nanosheets and honey was incorporated into chitosan hydrogel to form a mixed 0, 2 and 3 dimensional composite. The scanning electron microscopic (SEM) images of MXene nanosheets depict proper delamination of uniform layers with approximately 200 nm wall-to-wall interlayer spacing (Fig. 1a and b). The transmission electron microscopic (TEM) micrographs revealed the architectural etching of MXene nanosheets morphology in uniform colloidal suspension (Fig. 1c). The crystal lattice of Ti3C2 MXene
Second, MXene is now considered as a booming carbon-based 2D material which is biocompatible, conductive in nature, and has antibacterial properties (Huang et al., 2018). We recently reported that Ti3C2 MXene based biomaterials possess intrinsic anti-inflammatory and immunomodulatory properties. In this study, we found that Ti3C2 quantum dots selectively suppress activation of CD4+IFN-γ+ T-lymphocytes and promote expansion of immunosuppressive CD4+CD25+FoxP3+ regulatory T. Furthermore, we reported that MXene quantum dots were able to downregulate the levels of inflammatory cytokines TNF-α and IFN-γ, also there was an increasing trend in the levels of anti-inflammatory cytokine IL-10 (Rafieerad et al., 2019; Soleymaniha et al., 2019). Moreover, Ti3C2 MXene nanosheets with enhanced surface properties have been found to be electrically conductive and possess autofluorescence properties (Wang et al., 2017). Third, the application of chitosan based 3D polysaccharide hydrogels and scaffolds in regenerative medicine is widely reported (Suh and Matthew, 2000). Chitosan is a biocompatible, biodegradable and clinically accepted polymer. Therefore, addition of electrically conductive, anti-bacterial and anti-inflammatory agents can provide a wide avenue to biomedical applications of chitosan based 3D biomaterials. Furthermore, fabrication of a mixed-dimensional hydrogel composite has several advantages. For example, two-dimensional (2D) planar and well-defined geometry of MXene nanosheets will provide large surface area for cell attachment and growth. Also, addition of 2D carbide based material in a composite enhances its surface energy, hydrophilicity and conductivity (Zhang et al., 2018). Whereas, the 3D architecture of chitosan will mimic native tissue in the body. Also, biomaterials based 3D assemblies facilitate cellular interactions that 2
Journal of the Mechanical Behavior of Biomedical Materials 101 (2020) 103440
A. Rafieerad, et al.
Fig. 2. Physico-mechanical properties of the synthesized S-MXene hydrogels containing low molecular weight (L) and medium molecular weight (M) chitosan. (a, b) Swelling ability and water absorption capacity of the hydrogels were measured by immersing the scaffold in DMEM for 1 h (yellow) and 24 h (brown). (c) Degradation rate of the composite hydrogels (S-MXene L-brown; S-MXene M-yellow) in lysozyme containing medium was measured for 96 h at 37 °C. (d) Electrical resistivity (yellow) and conductivity (brown) of aqueous Ti3C2 MXene, S-MXene L and S-MXene M hydrogels was assessed by measuring resistivity of the composites by digital multimeter. (e-n) Long-term stability of the crosslinked S-MXene L (e,g,i,j) and S-MXene M (f,h,k,l) hydrogels at 37 °C was determined by incubating the biocomposites with or without culture medium. The hydrogel architecture was intact for 2 weeks in the presence or absence of medium at 37 °C. (m,n) Self-healing potential was measured by cutting the S-MXene hydrogels (L and M respectively) into two pieces and incubating these pieces together at 37 °C for 30 min. At the end of incubation period the hydrogel was able to heal itself. (o) Injectability of S-MXene L and M hydrogels (left to right) was tested using a 26 g needle and 1 ml syringe. (n = 3). 3
Journal of the Mechanical Behavior of Biomedical Materials 101 (2020) 103440
A. Rafieerad, et al.
Fig. 3. Assessment of autofluorescence of the hydrogel. MXene nanosheets and carbon dots present in honey naturally display autofluorescence properties, which can be exploited for imaging, tracking, biosensors related applications. (a-d) The autofluorescence of Ti3C2 MXene nanosheets (a,b) and S-MXene hydrogel (c,d) was detected by Cytation 5 imaging system at different excitation/emission wavelengths; Dapi (377 nm, 447 nm), GFP (469 nm, 525 nm), Texas Red (586 nm, 647 nm) and Cy5 (628 nm, 685 nm) as well as the combined (merged) images of all four filters. The MXene nanosheets and C-dots incorporated in chitosan architecture displayed autofluorescence at different wavelengths.
nanostructures into chitosan polymers can presumably limit the mobility of chitosan chains depending on the surface adsorption of functional groups, also it reduces the crystalline phase with weak crystal rings (Chaudhry and Mittal, 2013; Justin et al., 2015). The physicochemical properties of the S-MXene composite including swelling ability, degradation rate, stability, electrical conductivity and self-healing nature were measured as described in supplementary methods. Generally, addition of carbon-based nanocomposites into the structure of polymers e.g. chitosan, potentially enhances the mechanical properties (Baradaran et al., 2014; Lipatov et al., 2018; Qiu and Wang, 2011; Young et al., 2018; Zhang et al., 2016). On the other hand, the molecular weight of chitosan hydrogel is also reported to have a direct effect on mechanical stability of the composite (Huei and Hwa, 1996). To this end, in the current study, we fabricated S-MXene hydrogel with low molecular weight chitosan (S-MXene L) as well as medium molecular weight S-MXene (S-MXene M). We compared the physico-mechanical behavior of both S-MXene L and S-MXene M hydrogels. There were no significant changes found in the swelling ability, biodegradation rate, stability, self-healing and injectability of these two biocomposites. Therefore, presence of MXene nanosheets preserves mechanical properties of the chitosan based composite. We found that S-MXene hydrogels displayed excellent swelling ability (Fig. 2a and b). It is biodegradable, self-healing and electrically conductive in nature (Fig. 2c–p). It is already reported that Ti3C2 MXene as well as honey (due to the presence of C-Dots) display autofluorescence properties, which can be exploited for imaging, tracking, biosensors related applications (Zhou et al., 2017). In the current study, the MXene nanosheets separately as well as S-MXene hydrogel displayed excellent autofluorescence at different excitation/emission wavelengths; Dapi (377 nm, 447 nm), GFP (469 nm, 525 nm), Texas Red (586 nm,
nanosheets confirmed d-spacing around 0.220 nm corresponding to MXene facets. The EDS analysis further confirmed the removal of Al from MAX phase with well-defined elemental compositions of multilayer Ti3C2 and –NH group (Fig. 1d; Supplementary Fig. S1). Our data also demonstrate a uniform distribution of carbon (C) and titanium (Ti) elements with negligible traces of Al (Fig. 1d; Supplementary Fig. S1). Additionally, the structure of Ti3C2 MXene material also includes notable amounts of nitrogen (N), fluorine (F) and oxygen (O) following exfoliation with HF (Halim et al., 2016). The SEM images of S-MXene hydrogel composite confirmed highly-porous crosslinked networks composite with the pore size in the range of 2.62 μm–69.2 μm (Fig. 1e and f). The porous network of a biomaterial is important to exhibit sufficient swelling kinetic and facilitate oxygen and nutrients transfer to the cells. The EDS analysis detected uniform distribution of different elements of honey and MXene in the hydrogel (Fig. 1g and h; Supplementary Fig. S2). The high-resolution TEM and selected area electron diffraction (SAED) micrographs confirmed clear multi-dimensional crystalline patterns of S-MXene hydrogel composite (Fig. 1i–k). The fast fourier transform (FFT) images revealed the amorphous phase transmission of crystal lattices at an approximate range of 0.200–0.330 nm (Fig. 1l; Supplementary Fig. S3). It is important to note that the crystal phase in the structure of S-MXene hydrogel was attributed to crystalline lattice of Ti3C2 MXene nanosheets. Our results indicate that, the lateral d-spacing corresponds to Ti3C2 MXene (1 0 5) and Ti atom (1 1 2 0) facets (Li et al., 2017; Luo et al., 2017; Mashtalir et al., 2013; Xu et al., 2018). Our TEM results of S-MXene hydrogel also show d-spacing lattice of around 2.0 Å and 3.3 Å for MXene and carbon dots respectively (Supplementary Fig. S3), which is in agreement with the reported range in literature. Furthermore, studies on the crystal analysis of chitosan based composites found that addition of carbon 4
Journal of the Mechanical Behavior of Biomedical Materials 101 (2020) 103440
A. Rafieerad, et al.
Fig. 4. Biocompatibility of the S-MXene hydrogel with (a) rat bone marrow derived mesenchymal stem cells (MSCs) and (b) human iPSCs derived cardiomyocytes was assessed by determining cell viability using a fluorescent dye Calcein AM. The green fluorescent Calcein AM will stain only live cells. Dapi was used to stain nucleus. (c) MSCs after 24 h and (d) cardiomyocytes after 48 h of culture in the presence of S-MXene hydrogel showed excellent viability. Therefore, the hydrogel is compatible with both cell types. Interestingly, we also found a statistically significant increase in the number of live cells in case of iPSC derived cardiomyocytes compared to control group. *p < 0.05 compared to control group. (n = 3–6). Data are expressed as mean ± SD.
cardiomyocytes and neurons are reported to worsen the disease pathophysiology during cardiovascular and neurological disorders. It is therefore conceivable that a mixed-dimensional- Ti3C2-MXene-honeychitosan biocomposite might offer additional benefits over a chitosan based hydrogel or scaffold when applied for tissue regeneration. Therefore, a multifunctional electrically conductive novel S-MXene hydrogel, which is compatible with different types of stem cells, and exert inherent antibacterial and anti-inflammatory properties, may have a great potential in facilitating tissue repair in several degenerative diseases such as cardiovascular, neurological and autoimmune diseases.
647 nm), Cy5 (628 nm, 685 nm) (Fig. 3). Next, we assessed the biocompatibility of S-MXene hydrogel. The composite displayed no cytotoxicity toward rat bone marrow derived mesenchymal stem cells (MSCs) and human induced pluripotent stem cells (iPSCs) derived cardiomyocytes (Fig. 4a–d). Bone marrow derived MSCs and iPSCs are already being tested in animal studies and Phase I and II clinical trials for cardiac regeneration, neurological disorders, diabetes, orthopedic diseases and autoimmune disorders (Trounson and McDonald, 2015). One of the challenges in clinical translation of stem cell therapy is poor survival of transplanted cells in the recipient. In the current study, we found that the S-MXene hydrogel was not only safe for the stem cells, also it promoted survival of iPSCs-derived cardiomyocytes (4b,d). In our ongoing studies we are testing the role of Ti3C2 MXene based materials on differentiation potential of iPSCs to cardiomyocytes. The elevated inflammation is a hall mark in case of degenerative diseases. Also, bacterial infections are reported to be associated with the pathogenesis of autoimmune diseases, cardiovascular disorders and other degenerative diseases (El Kholy et al., 2015; Kivity et al., 2009). Furthermore, conduction abnormalities in electrical signals among
Acknowledgment This work was supported by research grants from Canadian Institutes of Health Research.
5
Journal of the Mechanical Behavior of Biomedical Materials 101 (2020) 103440
A. Rafieerad, et al.
Appendix A. Supplementary data
Luo, J., Zhang, W., Yuan, H., Jin, C., Zhang, L., Huang, H., Liang, C., Xia, Y., Zhang, J., Gan, Y., 2017. Pillared structure design of MXene with ultralarge interlayer spacing for high-performance lithium-ion capacitors. ACS Nano 11, 2459–2469. Mandani, S., Dey, D., Sharma, B., Sarma, T.K., 2017. Natural occurrence of fluorescent carbon dots in honey. Carbon 119, 569–572. Mashtalir, O., Naguib, M., Mochalin, V.N., Dall'Agnese, Y., Heon, M., Barsoum, M.W., Gogotsi, Y., 2013. Intercalation and delamination of layered carbides and carbonitrides. Nat. Commun. 4 1716. Minden-Birkenmaier, B.A., Meadows, M.B., Cherukuri, K., Smeltzer, M.P., Smith, R.A., Radic, M.Z., Bowlin, G.L., 2019. The effect of manuka honey on dHL-60 cytokine, chemokine, and matrix-degrading enzyme release under inflammatory conditions. Med one 4. Qiu, J., Wang, S., 2011. Enhancing polymer performance through graphene sheets. J. Appl. Polym. Sci. 119, 3670–3674. Rafieerad, A., Yan, W., Sequiera, G.L., Sareen, N., Abu‐El‐Rub, E., Moudgil, M., Dhingra, S., 2019. Application of Ti3C2 MXene quantum dots for immunomodulation and regenerative medicine. Adv. Healthc. Mater 1900569. Saravanan, S., Sareen, N., Abu-El-Rub, E., Ashour, H., Sequiera, G.L., Ammar, H.I., Gopinath, V., Shamaa, A.A., Sayed, S.S.E., Moudgil, M., 2018. Graphene oxide-gold nanosheets containing chitosan scaffold improves ventricular contractility and function after implantation into infarcted heart. Sci. Rep. 8 15069. Soleymaniha, M., Shahbazi, M.A., Rafieerad, A.R., Maleki, A., Amiri, A., 2019. Promoting role of MXene nanosheets in biomedical sciences: therapeutic and biosensing innovations. Adv. Healthc. Mater. 8 1801137. Srimuk, P., Kaasik, F., Krüner, B., Tolosa, A., Fleischmann, S., Jäckel, N., Tekeli, M.C., Aslan, M., Suss, M.E., Presser, V., 2016. MXene as a novel intercalation-type pseudocapacitive cathode and anode for capacitive deionization. J. Mater. Chem. 4, 18265–18271. Suh, J.-K.F., Matthew, H.W., 2000. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. Biomaterials 21, 2589–2598. Tonks, A.J., Cooper, R., Jones, K., Blair, S., Parton, J., Tonks, A., 2003. Honey stimulates inflammatory cytokine production from monocytes. Cytokine 21, 242–247. Trounson, A., McDonald, C., 2015. Stem cell therapies in clinical trials: progress and challenges. Cell stem cell 17, 11–22. Wang, Z., Xuan, J., Zhao, Z., Li, Q., Geng, F., 2017. Versatile cutting method for producing fluorescent ultrasmall MXene sheets. ACS Nano 11, 11559–11565. Xu, Q., Ding, L., Wen, Y., Yang, W., Zhou, H., Chen, X., Street, J., Zhou, A., Ong, W.-J., Li, N., 2018. High photoluminescence quantum yield of 18.7% by using nitrogen-doped Ti 3 C 2 MXene quantum dots. J. Mater. Chem. C 6, 6360–6369. Young, R.J., Liu, M., Kinloch, I.A., Li, S., Zhao, X., Vallés, C., Papageorgiou, D.G., 2018. The mechanics of reinforcement of polymers by graphene nanoplatelets. Compos. Sci. Technol. 154, 110–116. Zhang, H., Wang, L., Chen, Q., Li, P., Zhou, A., Cao, X., Hu, Q., 2016. Preparation, mechanical and anti-friction performance of MXene/polymer composites. Mater. Des. 92, 682–689. Zhang, Y.-Z., Lee, K.H., Anjum, D.H., Sougrat, R., Jiang, Q., Kim, H., Alshareef, H.N., 2018. MXenes stretch hydrogel sensor performance to new limits. Science Advances 4, eaat0098. Zhou, L., Wu, F., Yu, J., Deng, Q., Zhang, F., Wang, G., 2017. Titanium carbide (Ti3C2Tx) MXene: a novel precursor to amphiphilic carbide-derived graphene quantum dots for fluorescent ink, light-emitting composite and bioimaging. Carbon 118, 50–57.
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jmbbm.2019.103440. Author contributions A.R, A.A, and S.D. conceptualized the study; A.R, GLS, P.K, A.A and SD designed the experiments; A.R, GLS, W.Y and P.K carried out experiments and acquired the data; A.R, GLS, P.K and SD interpreted the data, carried out data analysis and drafted the manuscript. Conflicts of interest Authors declare no competing interests. References Baradaran, S., Moghaddam, E., Basirun, W.J., Mehrali, M., Sookhakian, M., Hamdi, M., Moghaddam, M.N., Alias, Y., 2014. Mechanical properties and biomedical applications of a nanotube hydroxyapatite-reduced graphene oxide composite. Carbon 69, 32–45. Chaudhry, A., Mittal, V., 2013. High‐density polyethylene nanocomposites using masterbatches of chlorinated polyethylene/graphene oxide. Polym. Eng. Sci. 53, 78–88. El Kholy, K., Genco, R.J., Van Dyke, T.E., 2015. Oral infections and cardiovascular disease. Trends Endocrinol. Metab. 26, 315–321. Halim, J., Cook, K.M., Naguib, M., Eklund, P., Gogotsi, Y., Rosen, J., Barsoum, M.W., 2016. X-ray photoelectron spectroscopy of select multi-layered transition metal carbides (MXenes). Appl. Surf. Sci. 362, 406–417. Huang, K., Li, Z., Lin, J., Han, G., Huang, P., 2018. Two-dimensional Transition Metal Carbides and Nitrides (MXenes) for Biomedical Applications. Chemical Society Reviews. Huei, C.R., Hwa, H.-D., 1996. Effect of molecular weight of chitosan with the same degree of deacetylation on the thermal, mechanical, and permeability properties of the prepared membrane. Carbohydr. Polym. 29, 353–358. Justin, R., Román, S., Chen, D., Tao, K., Geng, X., Grant, R.T., MacNeil, S., Sun, K., Chen, B., 2015. Biodegradable and conductive chitosan–graphene quantum dot nanocomposite microneedles for delivery of both small and large molecular weight therapeutics. RSC Adv. 5, 51934–51946. Kivity, S., Agmon-Levin, N., Blank, M., Shoenfeld, Y., 2009. Infections and autoimmunity–friends or foes? Trends Immunol. 30, 409–414. Li, R., Zhang, L., Shi, L., Wang, P., 2017. MXene Ti3C2: an effective 2D light-to-heat conversion material. ACS Nano 11, 3752–3759. Lipatov, A., Lu, H., Alhabeb, M., Anasori, B., Gruverman, A., Gogotsi, Y., Sinitskii, A., 2018. Elastic properties of 2D Ti3C2Tx MXene monolayers and bilayers. Science Advances 4, eaat0491.
6