Bolstered cell proliferation by supramolecular assemblies of biodegradable biocompatible block copolymers

摘要

Introduction: An amphiphilic diblock copolymer comprising poly(ethylene glycol) (PEG) and poly(I-lactide) (PLLA) (PEG-b-PLLA) typically forms spherical micelles (Fig. 1B, D), and thus has been studied as a drug delivery vehicle for a long time. Recently, Kim et al., have found that supramolecular nanotubes are formed by introduction of a mesogen-like structure consisting of (thio)urea and benzylbenzamide (4UMBA) into the block junction of PEG and PLLA (Fig. 1 A, C). Then we prepared the 4UMBA containing block copolymers comprising PEG and poly(ε-caprolactone) (PCL) or poly(trimethy1ene carbonate) (PTMC) to vary the nanoshape, resulting in long nanofibers with a micron length for PCL and uniformly dispersed nanospheres for PTMC. Figure 1. The nature of these nano self-assemblies with biocompatibility and biodegradability is beneficial for not only drug delivery but also scaffold that mimics hierarchically self-assembling collagen fibers in extracellular matrix (ECM). Therefore, in this study, we examined these self-assembling nanostructures as an additive for cell culture, subsequently developing higher order structure like ECM by 'concerted' self-assembly with cells. Materials and Methods: All block copolymers were prepared by ring-opening polymerization of cyclic monomers as described elsewhere. For the 4UMBA-containing polymers, PEG_(5k)-4UMBA-OH prepared as previously reported was used as an initiator. MeO-PEG_(5k)-OH is used for synthesizing the control diblock copolymers (Fig. 2). Then, each block copolymer was dispersed in milli-Q water (1.0%) and diluted with α-MEM culture media to formulate the 0.05% polymer containing media. Normal human dermal fibroblast (NHDF, P = 9) was seeded on poly(ethylene terephthalate) (PET) substrates in a 24-well plate with 5.0 × 10~3 cells/cm~2 and incubated at 37C with 5% CO_2 for 24 h. Afterwards, the culture media was changed to the polymer containing media and incubated under the same condition. At time points of 6,12,24, and 48 h, cells were observed by phase-contrast microscopy (Fig. 1G-I). Figure 2. Results: All block copolymers were successfully prepared, and the molecular weight of each block was well controlled to around 5,000. In milli-Q water (0.1 %), PEG-4UMBA-PLLA formed tubular or cylindrical assemblies (Fig. 1C), and PEG-4UMBA-PCL showed much larger fiber structure (Fig. 1F). No anisotropic assemblies were confirmed for any other polymers. In addition, PEG-4UMBA-PLLA constructed fine network structure in PBS buffer (1.0%, Fig. 1E). After 6 hours of cell culture, only PEG-4UMBA-PLLA influenced the cell growth behavior (Fig. 1H). The number and size of cells in the view are obviously larger compared to those for PEG-b-PLLA (Fig. 1G) and blank sample (Fig. 11), indicating that nanoshape could stimulate cell growth as previously reported in supramolecular antimicrobials. The cell growth was almost the same in the case of other polymers including PEG-4UMBA-PCL. Even after 48 h of incubation, the cell growth seemed to differ little between the polymer containing media and blank (Fig. 1J), except PEG-4UMBA-PLLA. Conclusion: These results are still preliminary and late-breaking. The nanostructure of polymers in the culture media and location of the assemblies (inside vs outside of cells) are still being investigated. Nevertheless, this is the first report of the bolstered cell growth stimulated by supramolecular assemblies of synthetic polymers.