Effect of Anisotropic Aggregates Comprising Pegylated Block Copolymers on Cell Growth and Aggregation

Abstract

In recent years, effects of shape of nanomaterials on biological response has attracted attention. For example, rod or fibrous polymeric micelles show a longer circulation time in blood and preferential accumulation to tumor cells than a spherical counterpart.1) On the other hand, fibrous cationic molecular assemblies exhibit higher or/and broader spectrum of antimicrobial activity than a spherical counterpart2). In addition, nano/micro-topology of substrates for tissue engineering is now recognized as a tool of regulating cellular responses such as adhesion, extension, aggregation, and differentiation. However, there have not been active systems to control such cellular responses by shape of molecular assemblies. Previously, we have developed amphiphilic pegylated diblock copolymers conjugating poly(L-lactide) (PLLA), poly(e-caprolactone) (PCL), or poly(trimethylene carbonate) (PTMC), forming anisotropic aggregates in water by introduction of a supramolecular assembling motif in the block junction. The size and length of the anisotropic aggregates are varied depending on the type of polymer used in hydrophobic segments. In this context, we explore cell culture of normal human dermal fibroblasts (NHDFs) in the presence of these anisotropic rod/fiber-like aggregates to evaluate the effect of shape of aggregates in culture media on cellular responses. The polymer aggregates were formulated by membrane dialysis. Typically, the block copolymer (40 mg) was dissolved in DMF (1 mL) and stirred overnight at room temperature. Nile red (NR) as a fluorescent probe was dissolved in DMF to have the concentration of 0.02 mg/mL. Then, the DMF solution of NR (1 mL) was added to the block copolymer solution and stirred for 2 hours. After that, the solution was transferred to a dialysis tube (Spectra/Por®7 Dialysis Membrane) with molecular cut off (MWCO) of 2,000 and dialyzed against ultrapure water for two days. Finally, the suspension was diluted to have the polymer concentration of 10 mg/mL (1.0 wt%) NHDFs were seeded on poly(ethylene terephthalate) (PET) plates embedded in a 24-well polystyrene plate at a cell density of 0.5 × 104 cells/cm2 in a cell culture medium (950 µL). The polymer suspension (50 µL) was then added to adjust the final polymer concentration to 0.05 wt%. The cells were incubated at 37 °C in 5% CO2 for 24 h and 48 h. At a certain time of incubation, cells were washed and stained with phalloidin and DAPI to visualize actin and nuclei in the observation by a confocal laser scanning microscope (CLSM). Furthermore, as three-dimension cell culture, NHDFs were first treated with CellTrackerTM to stain cytoplasm. Then, the cell suspension with a density of 1.6 × 104 cells/well including 0.05 wt% of the polymer aggregates was loaded in PrimeSurface\U0001f12c a round-bottom 96-well plates. The cells were incubated at 37 °C in 5% CO2 for 2, 6, and 12 h. Afterwards, cells were washed, stained with DAPI, and observed on a CLSM and a phase-contrast microscope. References 1)D. Li et al., Adv. Funct. Mater., 2016, 26, 66–79. 2)K.Fukushima et al., ACS Nano, 2012, 6, 9191-9199.