Chitosan/hyaluronic acid (HA) nanoparticles can be used to deliver an RNA/DNA cargo to cells overexpressing HA receptors such as CD44. For these systems, unequivocal links have not been established yet between chitosan macromolecular (molecular weight; degree of deacetylation, i.e., charge density) and nanoparticle variables (complexation strength, i.e., stability; nucleic acid protection; internalization rate) on one hand, and transfection efficiency on the other hand. Here, we have focused on the role of avidity on transfection efficiency in the CD44-expressing HCT-116 as a cellular model; we have employed two differently sized payloads (a large luciferase-encoding mRNA and a much smaller anti-Luc siRNA), and a small library of chitosans (variable molecular weight and degree of deactylation). The RNA avidity for chitosan showed-as expected-an inverse relationship: higher avidity-higher polyplex stability-lower transfection efficiency. The avidity of chitosan for RNA appears to lead to opposite effects: higher avidity-higher polyplex stability but also higher transfection efficiency. Surprisingly, the best transfecting particles were those with the lowest propensity for RNA release, although this might be a misleading relationship: for example, the same macromolecular parameters that increase avidity can also boost chitosan's endosomolytic activity, with a strong enhancement in transfection. The performance of these nonviral vectors appears therefore difficult to predict simply on the basis of carrier- or payload-related variables, and a more holistic consideration of the journey of the nanoparticle, from cell uptake to cytosolic bioavailability of payload, is needed. It is also noteworthy that the nanoparticles used in this study showed optimal performance under slightly acidic conditions (pH 6.4), which is promising for applications in a tumoral extracellular environment. It is also worth pointing out that under these conditions we have for the first time successfully delivered mRNA with chitosan/HA nanoparticles.

CD44 is a potentially rewarding target in cancer therapy, although its mechanisms of ligand binding and internalization are still poorly understood. In this study, we have established quantitative relationships between CD44 expression in differently polarized macrophages (M0, M1, and M2-polarized THP-1 human macrophages) and the uptake of hyaluronic acid (HA)-based materials, which are potentially usable for CD44 targeting. We have validated a robust method for macrophage polarization, which sequentially uses differentiating and polarizing factors, and allows to show that CD44 expression depends on polarization (M1 > M0 ≥ M2). It is noteworthy that THP-1 M2 expressed CD44v6, suggesting their suitability as a model of tumor-associated macrophages. In the uptake of HA, both as a soluble polymer and in the form of (siRNA-loaded) nanoparticles, CD44 expression correlated positively with binding, but negatively with internalization. Counterintuitively, it appears that a higher presence of CD44 (in M1) allows a more efficient capture of HA materials, but a lower expression (in M2) is conducive to better internalization. Although possibly cell-specific, this unexpected relationship indicates that the common paradigm "higher CD44 expression = better targetability" is too simplistic; mechanistic details of both receptor presentation and association still need to be elucidated for a predictable targeting behavior.

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Measuring the viscoelastic behavior of highly hydrated biological materials is challenging because of their intrinsic softness and labile nature. In these materials, it is difficult to avoid prestress and therefore to establish precise initial stress and strain conditions for lumped parameter estimation using creep or stress-relaxation (SR) tests. We describe a method ( or epsilon dot method) for deriving the viscoelastic parameters of soft hydrated biomaterials which avoids prestress and can be used to rapidly test degradable samples. Standard mechanical tests are first performed compressing samples using different strain rates. The dataset obtained is then analyzed to mathematically derive the material's viscoelastic parameters. In this work a stable elastomer, polydimethylsiloxane, and a labile hydrogel, gelatin, were first tested using the, in parallel SR was used to compare lumped parameter estimation. After demonstrating that the elastic parameters are equivalent and that the estimation of short-time constants is more precise using the proposed method, the viscoelastic behavior of porcine liver was investigated using this approach. The results show that the constitutive parameters of hepatic tissue can be quickly quantified without the application of any prestress and before the onset of time-dependent degradation phenomena. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3352–3360, 2014