Cellular response to phase-separated blends of tyrosine-derived polycarbonates

Abstract

Two-dimensional thin films consisting of homopolymer and discrete compositional blends of tyrosine-derived polycarbonates were prepared and characterized in an effort to elucidate the nature of different cell responses that were measured in vitro. The structurally similar blends were found to phase separate after annealing with domain sizes dependent on the overall composition. The thin polymer films were characterized with the use of atomic force microscopy (AFM), water contact angles, and time-of-flight secondary ion mass spectrometry (TOF-SIMS) and significant changes in roughness were measured following the annealing process. Genetic expression profiles of interleukin-1beta and fibronectin in MC3T3-E1 osteoblasts and RAW 264.7 murine macrophages were measured at several time points, demonstrating the time and composition-dependent nature of the cell responses. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) depicted upregulation of the fibronectin gene copy numbers in each of the blends relative to the homopolymers. Moreover, the interleukin-1beta expression profile was found to be compositionally dependent. The data suggest strongly that optimal composition and processing conditions can significantly affect the acute inflammatory and extracellular matrix production responses.

Figure 1

Synthetic scheme and chemical structure desaminotyrosyl-tyrosine alkyl esters and the resulting polycarbonates. The pendent R groups of the polycarbonates reported in this article consist of ethyl, butyl, hexyl, and octyl esters, respectively. The corresponding polymers are referred to as poly(DTE carbonate), poly(DTB carbonate), poly(DTH carbonate), and poly(DTO carbonate).

Figure 2

From left to right in the five 5 × 5-μm AFM images shown above, the amount of DTO is increasing. The discrete DTE/DTO blends form phase-separated domains under in vacuo, 105°C annealing conditions. The size and spacing of the domains are tunable by varying the composition of the blend. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 3

Negative-ion TOF-SIMS of the tyrosine-derived homopolymers and the respective blends showing the characteristic peaks for DTE (191) and DTO (275) and the chemical structures for the common ion fragments first identified by Belu et al. show that both materials are present at the surface in the phase-separated blends.

Figure 4

(A) Gene copy numbers of IL-1β and TNF-α in RAW 264.7 macrophages after 24 h of exposure. (B) Relative differences in actin, fibronectin, and collagen I gene copy numbers in MC3T3-E1 osteoblasts for each of the tyrosine-derived polycarbonates (DTE, DTB, DTH, and DTO, respectively), TCPS, and PCL after 3 h of incubation. Error bars are representative of one standard deviation from the mean of triplicate samples harvested from a single population of cells, and are the estimate of the standard uncertainties. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 5

Depicts gene copy numbers of interleukin-1β (IL-1β) (A and C), and fibronectin (B and D) after 24 h of exposure on the respective surface for RAW 264.7 macrophages (A and B) and MC3T3 E1 bone osteoblasts (C and D). Error bars are representative of one standard deviation from the mean of triplicate samples harvested from a single population of cells, and are the estimate of the standard uncertainties. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 6

Immunofluorescent staining for (A) actin, (C) vinculin, (B) overlays of MC3T3-E1 osteoblasts, and (D) RAW 264.7 macrophages, showing the cytoskeleton and focal adhesion contact formation 16 h after seeding on each of the respective homopolymers and blends. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]