br Introduction br Multicellular cancer spheroids
Multicellular cancer spheroids (CSs), comprising a compact three-dimensional spheroidal aggregation of cancer cells, inherit much more characteristics properties of solid tumors, such as extracellular matrix deposition between the cells, strong cell-cell junctions and gradients in nutrient concentration, therefore potentially be of great significance to studies in cancer biology, metastasis and invasion [1–4]. For example, CSs have been used in studies of the role of mechanical stresses on tumor progression [5–7] and the invasive behavior of tumors [8–10]. Importantly, CSs are used for the evaluation of the efficiency of antic-ancer drugs [11–13]. Up to now, CSs have been prepared by using a variety of techniques or devices such as the growth of cells on non-adhesive surfaces or in suspension [14–16], cell implantation in a prefabricated polymer scaffold [17–19], or culture cells within hy-drogel platform [20–24]. However, in vitro growth of CSs from in-dividual cells is challenging, as some types of cancer cells do not grow in vitro. In addition, tumor cells from certain cell lines could only form loose, irregular UNC1999 rather than CSs with physiological barriers and pathophysiological gradients, which failed to mimic in vivo solid tumor in these aspects. Therefore, the development the novel cell
∗ Corresponding author.
culture method that permits reversible control between monolayer and spheroid cultures is still in urgent need.
On the other hand, in order to quantitatively characterize the CSs encapsulated in hydrogels at the cellular and molecular level, the post-growth release of CSs from hydrogel scaffolds is highly desirable [3,14,20]. Many stimuli-responsive hydrogels rooted in the use of modified substrates have been utilized [25–27], where CSs growth and release can be regulated in response to external stimuli, e.g., tempera-ture [28–31], enzyme [15,32–34], pH [35–37], or light [38–43]. Each one of these strategies has their advantages and limitations. For ther-moresponsive hydrogels, they require the precise control of the surface temperature of the device to achieve CS encapsulation, growth and release, thus, additional equipment to control the temperature is re-quired and limits the ability to commercially scale these devices. Moreover, the use of enzymes or light responds hydrogels may be harmful to the encapsulated cells or affect CSs integrity [39,44]. Therefore, it is necessary and important to explore and synthesise a new response type of hydrogels that not only facilitate viable CSs growth and release but also keep CSs integrity.
Herein, we synthesize anions reversibly responsive luminescent nanocellulose hydrogels for the growth and subsequent release of CSs
Available online 15 December 2018
Scheme 1. (A) Schematic illustration of the synthesis of Eu(III) complex-CMC, K-DPY-CMC, and CDEAC hydrogels. (B) Encapsulation, growth, and release of CSs from the CDEAC hydrogels.
(Scheme 1). Such hydrogel is made up of two parts. One part is Eu(III) 2-(2-aminobenzamido) benzoic acid complex functionalized carbox-ymethyl cellulose (CMC) (denote as Eu(III) complex-CMC). Another part is 2, 6-pyridinedicarboxylic acid functionalized CMC (denote as K-DPY-CMC). In our study, carboxymethyl cellulose was selected as the conjugate backbone based on its safety profile for use in parenteral formulations, the reasonable stability of the backbone in human phy-siological conditions [45–47]. The resultant Eu(III) complex-CMC and K-DPY-CMC individually form colloidally stable suspensions (or solu-tions) but assemble into coordinately cross-linked red fluorescence hydrogel with entrapped MCF-7 breast cancer cells, when these two kinds of CMCs mixed together. The formed hydrogel (denote as CDEAC) which can be used as a replacement of Matrigel as it allows the culture of CSs. Because ClO− is easily coordinated with Eu ion, leading to cross-linked cellulose cleavage, thus, when the ClO− was added to the above multicellular CSs-laden hydrogel, the dissociated hydrogel releases CSs, accompanying by the fluorescent quenching. Moreover, treating the dissociated hydrogel with SCN− could restore the cross-linked cellu-lose. Additionally, the dissociation-adhesion transition of the hydrogel can be induced via the regulation of ClO−/SCN−.
2. Methods and materials
Carboxymethyl cellulose (CMC, Mw ∼250,000, Aldrich), hydro-chloric acid (HCl, Sigma), N,N-dimethylformamide (DMF, 99%, Aldrich), 1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide HCl (EDC HCl, Sigma-Aldrich), N-hydroxysuccinimide (NHS, Sigma-Aldrich), 2-(2-aminobenzamido) benzoic acid (AMBA, 98%, Aldrich), 2,6-di-methyl-pyridin-4-ylamine (DPY, Aldrich), Potassium permanganate (KMnO4, 98%, Alfa Aesar). All chemicals, unless specified, were used as received without further purification.