Phosphorylcholine-Modified Chitosan Films as Effective Promoters of Cell Aggregation: Correlation Between the Films Properties and Cellular Response

Abstract

This study describes chitosan-phosphorylcholine (CH-PC) films able to support the formation of cell aggregates (spheroids), which are important for tissue engineering and pharmacological studies. The surface topography, charge, thickness, and rheology of CH-PC thin films were characterized by AFM, zeta-potential measurements, SPR spectroscopy, and QCM-D measurements. The CH-PC films are highly hydrated gels, independently of the level of PC incorporation (15-40 mol-% PC/glucosamine units). QCM-D studies established that the amount of fibrinogen adsorbed on CH-PC films decreased with increasing PC content. CH-PC surfaces underwent a transition from moderately cell-adhesive (CH-PC15) to non-adhesive (CH-PC40). Optical micrographs of HUVEC and MCF-7 cell lines cultured on CH-PC surfaces showed that they form spheroids on CH-PC25 and CH-PC40 films.

Spreading of Cell Aggregates on Zwitterion-Modified Chitosan Films

“…The thicknesses of the CH-SB films (∼20 nm) derived from the Voigt model increase slightly with increasing SB content, as observed also in the case of CH-PC previously reported (film thickness of ∼10 nm for CH-PC15 and ∼15 nm for CH-PC40). 18 The thin CH-SB and CH-PC20 films prepared here on glass substrates are considered to be rigid surfaces; hence in the following, we will neglect the effect of the film rheology on the adhesion of cells. 33 In a previous study of CH-PC films by takeoff angle-dependent XPS analysis of vacuum-dried CH-PC films, coupled with a QCM-D analysis of the rehydrated films, we established that the vacuum-exposed CH-PC film surface is enriched in PC groups, compared to the film bulk.…”

“…Moreover, the PC groups remain located preferentially on the water/film interface upon rehydration, due to their hydrophilic nature. 18,36 By analogy, we surmise that during deposition of CH-SB on the substrates, the hydrophilic SB groups preferably orient themselves toward the water/polymer interface rather than being embedded within the polymer.…”

“…Chitosan (degree of deacetylation, DDA ∼ 75%), fibronectin, magnesium nitrate, sodium hydroxide, sodium acetate, 3-dimethylaminopropane 1, 2-diol, sodium periodate, 1, 3-propane-sultone, and acetic acid were purchased from Aldrich Chemical Co. Streptomycin and penicillin were purchased from Gibco BRL. Deacetylated chitosan (2 mol % N -acetylamine groups, DDA ∼ 98%) 17 and phosphorylcholine-chitosan (CH-PC20) 18 were prepared as reported previously. Spectra/Pore membranes (Spectrum) were employed for dialysis.…”

“…The clean sensors were kept overnight in a 1 mM 11-mercaptoundecanoic acid (MUA) solution in ethanol. 18 Subsequently, they were washed with ethanol and dried with nitrogen. The MUA-coated sensors were mounted in the QCM-D flow chamber and flushed with PB S (pH 7.4) until the baseline was stable.…”

“…We also observed that protein adsorption was low on all CH-PC films immersed in aqueous protein solutions and that the protein surface density decreased markedly with increasing PC content of the substrate. 18 Both human umbilical vein endothelial cells (HUVEC) and a human breast cancer cell line (MCF-7) adhered poorly on CH-PC films. Cells showed a marked tendency to aggregate on the substrates, 21 a phenomenon typical for cohesive cells seeded on nonfouling substrates.…”