Cell-Responsive Shape Memory Polymers

Abstract

Recent decades have seen substantial interest in the development and application of biocompatible shape memory polymers (SMPs), a class of "smart materials" that can respond to external stimuli. Although many studies have used SMP platforms triggered by thermal or photothermal events to study cell mechanobiology, SMPs triggered by cell activity have not yet been demonstrated. In a previous work, we developed an SMP that can respond directly to enzymatic activity. Here, our goal was to build on that work by demonstrating enzymatic triggering of an SMP in response to the presence of enzyme-secreting human cells. To achieve this phenomenon, poly(ε-caprolactone) (PCL) and Pellethane were dual electrospun to form a fiber mat, where PCL acted as a shape-fixing component that is labile to lipase, an enzyme secreted by multiple cell types including HepG2 (human hepatic cancer) cells, and Pellethane acted as a shape memory component that is enzymatically stable. Cell-responsive shape memory performance and cytocompatibility were quantitatively and qualitatively analyzed by thermal analysis (thermal gravimetric analysis and differential scanning calorimetry), surface morphology analysis (scanning electron microscopy), and by incubation with HepG2 cells in the presence or absence of heparin (an anticoagulant drug present in the human liver that increases the secretion of hepatic lipase). The results characterize the shape-memory functionality of the material and demonstrate successful cell-responsive shape recovery with greater than 90% cell viability. Collectively, the results provide the first demonstration of a cytocompatible SMP responding to a trigger that is cellular in origin.

Keywords: Pellethane; cell-responsive polymers; cytocompatibility; poly(ε-caprolactone); shape-memory polymers.

Scheme 1. Approach for Demonstrating Enzymatic Triggering of a Shape-Memory Polymer in Direct Response to the Presence of Enzyme-Secreting Human Cells

Sample from the PCL–PEL 400 group (text for details), with the illustration of the mechanism (top row), and macro- (middle) and micro- (bottom) views as the sample undergoes the shape-memory cycle.

Figure 1

Content percentage was calculated via DSC by dividing the enthalpy change of PCL (approximately 41.5 J/g) by the enthalpy change of the PCL–Pellethane fiber (approximately 8.5 J/g). The calculated composition of the representative sample being shown is 20.48%. Only content percentages close (±5%) to the predicted values were used in subsequent experiments.

Figure 2

(A) Extracellular lipase concentration in the medium of cultured HepG2 cells increased over time, regardless of whether or not the cells were treated with heparin, but, by day 7, cells treated with heparin had a significantly higher lipase concentration than cells without heparin treatment. (B) Intracellular lipase concentration of the cultured HepG2 cells decreased with time but was orders of magnitude higher than the extracellular lipase concentration. The relation between absorbance and lipase concentration can be found in the Supporting Information (Figure S1A,B) (n = 3, two-way ANOVA, followed by Holm–Sidak’s multiple comparisons test between groups. *p < 0.05, **p < 0.01).

Figure 3

Presence of heparin-treated cells led to recovery of the fiber structure to the as-spun state after 4 weeks of incubation (boxed image), while no significant recovery-related morphological change was found in cells without heparin treatment or in samples in PBS. PCL 3000 fibers showed no recovery-related morphological changes after 4 weeks of culture in PBS but became coarser over time and eventually showed a film-like morphology at 4 weeks in some areas of the fiber networks in the presence of cells. PEL fibers showed no morphological changes after 4 weeks of culture under any control or treatment conditions compared to the programmed ones. Unidentified small particles or binders (representative particles or binders are identified by arrows) were observed to be embedded in the fiber networks in the presence of cells. All 3000 rpm as-spun fibers showed an aligned orientation. The programmed PEL 3000 and PCL–PEL 3000 showed a more tortuous arrangement without preferential alignment compared to the as-spun samples. Representative particles (red) or binders (blue) are identified by arrows. Scale bar: 10 μm.

Figure 4

Shape recovery was successfully triggered by heparin-treated cells in random and aligned PCL–PEL fibers. Strain recovered during shape recovery over 4 weeks was calculated. To account for the non-shape-memory changes, such as curling, observed in the Pellethane controls, the strain change of the Pellethane controls (Figure 3A,B) was subtracted from that of the PCL–PEL experimental groups (Figure 3C,D) to determine the normalized shape-memory recovery (Figure 3E,F). The majority of strain recovery was completed by 2 weeks of incubation. Trends without statistical significance were observed in the strain change of the control groups: PBS and cells without heparin treatment (PBS and CELL; n = 3). One-way ANOVA was followed by Tukey’s post hoc test between groups. Markers indicating a significant difference (*p < 0.05, **p < 0.01) are for comparisons made against the PBS control.

Figure 5

Analysis of cell viability of HepG2 cells cultured on tissue culture plates with fiber composites for 7 d found no significant statistical differences in cell viability compared to control groups. All groups had a viability of 90% or greater over 7 d (n = 3, one-way ANOVA, followed by Tukey’s post hoc test between groups).

Figure 6

Live/dead micrographs of HepG2 cells cultured on tissue culture plates with fiber composite and noncomposite control showed cell proliferation over the period of incubation and few dead cells (red dots) in all groups, which revealed the indirect cytocompatibility of the fibers. Scale bar: 330 μm.