Stimuli-responsive polymers for rapid and reversible cell spheroid formation

摘要

Cell surface biomolecules govern numerous vital processes of the life cyde of cells. The ability to decorate the cell membrane with functional motifs has emerged as a powerful technique to manipulate various biological and unnatural functionalities for cell-based therapies. Particularly, cell surface engineering finds great interest in the construction of 3D cell/tumour spheroids and hybrid ensembles in the field of tissue engineering. For instance, cellular assemblies can establish an in vivo-like micro-environment and bridge the gap between 2D cell culture and tissue. In this study, we introduce two simple cell-interacting copolymers that trigger and control cell aggregation cascades in a fully reversible manner. A copolymer (P1, M_n 14100 Da) based on N-vinylpyrrolidone and 3-(acrylamido)phenylboronic acid (APBA) was used as a cis-diol reacting moiety to target cell membrane glycoprotein residues via diol-boronate ester formation, which can be out-competed by the addition of diol-rich compounds (e.g. glucose). A copolymer of di(ethylene glycol) methacrylate (DEGMA) and N-hydroxysuccinimide (P2, M_n 29800 Da), with a lower critical solution temperature (LCST) onset at sub-cell culture conditions, acted as a thermoresponsive targeting element of free primary amino groups on membrane proteins. Human dermal fibroblasts (HDF) and A549 (lung cancer) cells were incubated with P1 and P2 at different concentrations at room temperature. Both copolymers were found to induce rapid cell aggregation at low concentrations (200 μg/mL) in complete culture medium within 20 minutes (Fig.1). Fig.1. Representative microscopy images of cell aggregation in presence of P1 and P2. Various control experiments were then conducted to probe the specificity of the proposed mechanism. An increase of free glucose concentration in P1-cell suspension resulted in a gradual reduction of the cell aggregates. At the same time, incubation of APBA-free polymer with cells showed no aggregates formation. In a similar procedure, poly(DEGMA) and non-thermoresponsive PEG-based polymers were tested and no aggregation was observed. Hence, anchoring of P2 on the cell membrane concerted by temperature modulated coil-to-globule polymer transition resulted in the rapid formation of cellular clusters. To demonstrate the reversibility of the aggregation process, a thermal cycle was conducted by lowering of the temperature below the polymer's LCST. Furthermore, no toxicity was observed for P1 and P2 in both cell lines after 24 hours incubation period at high polymer concentrations (up to 500 μg/mL), as shown in Fig. 2. Fig. 2. Effect of polymer concentration on cell viability after 24 h incubation with P1 and P2 for both cell lines (mean ± SD obtained from triplicates, *p ≤ 0.05). These data inspired us to investigate the possibility of forming macroscopic "cell glues". Interestingly, upon mixing the polymers with cells (50 mg/mL) at room temperature we could form stable self-supporting gels. To conclude, we reported two simple copolymers for generic cell surface remodelling that induce rapid and reversible cell aggregation within minutes at minute polymer concentrations. The polymers could also form stable "cell-rich" gels at low concentrations under physiological conditions. We anticipate that this study will find uses in tissue regeneration and cell transplantation or as 3D tumour models.