Dual roles of hyaluronic acids in multilayer films capturing nanocarriers for drug-eluting coatings.

We developed hyaluronic acid (HA)-based multilayer films capturing polymeric nanocarriers (NCs) for drug delivery. The electrostatic interactions between positively charged linear polyethylene imines (LPEI) and negatively charged HAs are the main driving forces to form multilayers based on the layer-by-layer (LbL) deposition. NCs were easily incorporated within the multilayer film due to intra- and/or inter-hydrogen bonding among HA chains. The amount of NCs captured by the HA chains was varied by the ratio between HAs and NCs as well as the length (i.e., molecular weight) and absolute number density of HAs in solution. Biocompatibility of the NC-capturing HA multilayer films was tested with the human dermal fibroblast (HDF) culture. In addition, the controlled release of paclitaxel (PTX) from the HA multilayer films successfully led to the apoptosis of human aortic smooth muscle cells (hSMC) in vitro, implying that the NC-capturing HA multilayer films would be quite useful as drug-eluting stent systems to prevent the restenosis after surgery.

Nanoparticle-functionalized polymer platform for controlling metastatic cancer cell adhesion, shape, and motility.

Controlling and understanding the changes in metastatic cancer cell adhesion, shape, and motility are of paramount importance in cancer research, diagnosis, and treatment. Here, we used gold nanoparticles (AuNPs) as nanotopological structures and protein nanocluster forming substrates. Cell adhesion controlling proteins [in this case, fibronection (Fn) and ephrinB3] were modified to AuNPs, and these particles were then modified to the layer-by-layer (LbL) polymer surface that offers a handle for tuning surface charge and mechanical property of a cell-interfacing substrate. We found that metastatic cancer cell adhesion is affected by nanoparticle density on a surface, and ∼140 particles per 400 µm(2) (∼1.7 µm spacing between AuNPs) is optimal for effective metastatic cell adhesion. It was also shown that the AuNP surface density and protein nanoclustering on a spherical AuNP are controlling factors for the efficient interfacing and signaling of metastatic cancer cells. Importantly, the existence of nanotopological features (AuNPs in this case) is much more critical in inducing more dramatic changes in metastatic cell adhesion, protrusion, polarity, and motility than the presence of a cell adhesion protein, Fn, on the surface. Moreover, cell focal adhesion and motility-related paxillin clusters were heavily formed in cell lamellipodia and filopodia and high expression of phospho-paxillins were observed when the cells were cultured on either an AuNP or Fn-modified AuNP polymer surface. The ephrin signaling that results in the decreased expression of paxillin was found to be more effective when ephrins were modified to the AuNP surface than when ephrinB3 was directly attached to the polymer film. The overall trend for cell motility change is such that a nanoparticle-modified LbL surface induces higher cell motility and the AuNP modification to the LbL surface results in more pronounced change in cell motility than Fn or ephrin modification to the LbL surface.

Covalently bonded layer-by-layer assembly of multifunctional thin films based on activated esters.

We demonstrate that chemically stable, multifunctional polymer thin films can be obtained using the layer-by-layer (LbL) deposition based on covalent bonds between adsorbing chains. Poly(pentafluorophenyl-4-vinylbenzoate) (P1) or poly(pentafluorophenylacrylate) (P2) polymers were assembled with poly(allyl amine) (PAAm) to yield LbL multilayer films through amide bond formation by the reaction between activated esters of P1 or P2 and amine groups in PAAm, which was quantitatively monitored by Fourier transform infrared spectroscopy (FT-IR). It was found that the difference in the solubility of P1 and P2 against ethanol, which was used as the solvent for PAAm, during the LbL deposition yields different reaction conversion for the activated esters in either P1 or P2: the reaction conversion of P2 is higher than the conversion with P1. In addition, free (or unreacted) activated esters and amine groups remaining in the PAAm/P1 LbL film were further utilized for the incorporation of multiple functional materials (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid (EDANS) and Rhodamine B dyes in the present case) by post-treatments in order to further tailor the film properties. It was also demonstrated that the surface functional groups (activated esters) in the LbL films can also be utilized for surface patterning with one functional material, followed by functionalization with a second functional material during the post-treatment throughout the whole film. Finally, the PAAm/P1 and PAAm/P2 LbL films were shown to be quite stable in the extreme pH range, and free-standing films can easily be obtained by the treatment of the films with mild acidic conditions. The versatility of incorporating multiple functional materials into a single multilayer film as well as the excellent physicochemical stability of the covalently bonded multilayer free-standing films proves to be quite useful to design flexible and multifunctional thin film structures for many chemical and biological applications.

Tunable layer-by-layer polyelectrolyte platforms for comparative cell assays.

We developed a cell-based assay based on the spin-assisted layer-by-layer (LbL) assembled polyelectrolyte matrix platforms. Three types of human breast epithelial cell lines including normal cells (184B5), noncancerous fibrocystic disease cells (MCF 10F), and metastatic cancerous cells (CAMA-1) were cultured, analyzed, and compared in parallel on various LbL-assembled polymer films. Poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) electrolyte polymers were used as the basic building units to form various LbL polyelectrolyte matrices. The mechanical rigidity, surface charge, and biorecognition property of the LbL platforms were controlled by tailoring the LbL surface, thermal cross-linking, and protein modification. Cellular phenotypic changes in adhesion, proliferation, and morphology on these LbL films were characterized and analyzed for the three different cell types. Our analysis results indicate that the cellular phenotype can be controlled by taking advantage of different surface charge, mechanical property, and biological modification (i.e., fibronectin in this case) of the LbL multilayer platforms. Importantly, cell phenotypical quantification results show that the cell spreading area per cell and optical density are useful parameters in distinguishing metastatic cancer cells from normal or fibrocystic disease cells on these LbL films. These LbL-based cell assay platforms have a potential for the development of various disease diagnostic cell assays.

Effect of the layer-by-layer (LbL) deposition method on the surface morphology and wetting behavior of hydrophobically modified PEO and PAA LbL films.

We demonstrate that the surface morphology and surface-wetting behavior of layer-by-layer (LbL) films can be controlled using different deposition methods. Multilayer films based upon hydrogen-bonding interactions between hydrophobically modified poly(ethylene oxide) (HM-PEO) and poly(acrylic acid) (PAA) have been prepared using the dip- and spin-assisted LbL methods. A three-dimensional surface structure in the dip-assisted multilayer films appeared above a critical number of layer pairs owing to the formation of micelles of HM-PEO in its aqueous dipping solution. In the case of spin-assisted HM-PEO/PAA multilayer films, no such surface morphology development was observed, regardless of the layer pair number, owing to the limited rearrangement and aggregation of HM-PEO micelles during spin deposition. The contrasting surface morphologies of the dip- and spin-assisted LbL films have a remarkable effect on the wetting behavior of water droplets. The water contact angle of the dip-assisted HM-PEO/PAA LbL films reaches a maximum at an intermediate layer pair number, coinciding with the critical number of layer pairs for surface morphology development, and then decreases rapidly as the surface structure is evolved and amplified. In contrast, spin-assisted HM-PEO/PAA LbL films yield a nearly constant water contact angle due to the surface chemical composition and roughness that is uniform independent of layer pair number. We also demonstrate that the multilayer samples prepared using both the dip- and spin-assisted LbL methods were easily peeled away from any type of substrate to yield free-standing films; spin-assisted LbL films appeared transparent, while dip-assisted LbL films were translucent.