CD44 targeted delivery of siRNA by using HA-decorated nanotechnologies for KRAS silencing in cancer treatment.

KRAS is a small GTPase that regulates cell proliferation and survival. In tumors, the KRAS gene is mutated, and leading to unregulated tumor growth. Despite the recognized importance of KRAS in cancer, attempts to develop small molecule inhibitors have proved unsuccessful. An alternative strategy is gene silencing and the use of small nucleic acid sequences (e.g. siRNA, shRNA), has been reported to successfully downregulate KRAS. In this study we developed ternary nanocomplexes to deliver an anti-KRAS siRNA to colorectal cancer cells, exploiting the interaction of hyaluronic acid (HA) with CD44 as a means to achieve selective targeting of CD44-positive cancer cells. Two different polycations, poly(hexamethylene biguanide) and chitosan, were complexed with siRNA and coated with HA. Physico-chemical properties and stability of nanoparticles were characterized, including size, surface charge, and degree of siRNA protection. We demonstrate nanoparticle internalization (flow cytometry), siRNA cytosolic release (confocal microscopy) and KRAS silencing (RT-qPCR) in CD44+/KRAS+ colorectal cancer cell line, HCT-116. Further we demonstrate that the uptake of HA-decorated nanoparticles in cancer cells is higher when co-cultured with fibroblasts.

Colorectal tumor 3Din vitromodels: advantages of biofabrication for the recapitulation of early stages of tumour development.

The majority of cancer-relatedin vitrostudies are conducted on cell monolayers or spheroids. Although this approach has led to key discoveries, it still has a poor outcome in recapitulating the different stages of tumor development. The advent of novel three-dimensional (3D) systems and technological methods for their fabrication is set to improve the field, offering a more physiologically relevant and high throughputin vitrosystem for the study of tumor development and treatment. Here we describe the fabrication of alginate-based 3D models that recapitulate the early stages of colorectal cancer, tracking two of the main biomarkers for tumor development: CD44 and HIF-1α. We optimized the fabrication process to obtain alginate micro-beads with controlled size and stiffness, mimicking the early stages of colorectal cancer. Human colorectal HCT-116 cancer cells were encapsulated with controlled initial number, and cell viability and protein expression of said 3Din vitromodels was compared to that of current gold standards (cell monolayers and spheroids). Our results evidenced that encapsulated HCT-116 demonstrated a high viability, increase in stem-like cell populations (increased expression of CD44) and reduced hypoxic regions (lower HIF-1a expression) compared to spheroid cultures. In conclusion we show that our biofabricated system is a highly reproducible and easily accessible alternative to study cell behavior, allowing to better mimic the early stages of colorectal cancer in comparison to otherin vitromodels. The use of biofabricatedin vitromodels will improve the translatability of results, in particular when testing strategies for therapeutic intervention.

HisTOOLogy: an open-source tool for quantitative analysis of histological sections.

HisTOOLogy is an open-source software for the quantification of digital colour images of histological sections. The simple graphical user interface enables both expert and non-expert users to rapidly extract useful information from stained tissue sections. The software's main feature is a generalizable colour separation algorithm based on k-means clustering which accurately and reproducibly returns the amount of colour per unit area for any stain, thus allowing the quantification of tissue components. Here we describe HisTOOLogy's algorithms and graphical user interface structure, showing how it can be used to separate different dye colours in several classical stains. In addition, to demonstrate how the tool can be employed to obtain quantitative information on biological tissues, the effect of different hepatic tissue decellularization protocols on cell removal and matrix preservation was assessed through image analysis using HisTOOLogy and compared with conventional DNA and total protein content assays. HisTOOLogy's performance was also compared with ImageJ's colour deconvolution plug-in, demonstrating its advantages in terms of ease of use and speed of colour separation.

Viscoelastic characterisation of pig liver in unconfined compression.

Understanding and modelling liver biomechanics represents a significant challenge due to the complex nature of this organ. Unfortunately, there is no consensus on liver viscoelastic properties, and results are strongly dependent on sample type and status, adopted testing method, and testing conditions. Standard force-triggered tests (e.g. step response or dynamic mechanical tests) necessitate an initial contact between sample and testing apparatus, which may result in significant pre-stress to very soft and highly hydrated samples. In a previous study we proposed the epsilon dot method (ε̇M): a testing and analysis framework to address the drawbacks of standard mechanical tests. Focusing on ex-vivo unconfined bulk compressive tests, here we use both the ε̇M and dynamic mechanical analysis (DMA) to derive liver viscoelastic parameters in the region of small strains or the linear viscoelastic region (LVR). As liver samples were visibly deteriorated at the end of frequency sweep tests, a modified approach was adopted to reduce DMA testing times. This approach, termed step-reconstructed DMA (SRDMA), is based on dynamic measurements around specific frequencies and then reconstruction of liver behaviour in the entire frequency range of interest. The instantaneous elastic modulus obtained from SRDMA tests (2.65 ± 0.30 kPa) was significantly higher than that obtained with the ε̇M (2.04 ± 0.01 kPa). We show that the overestimation of stiffness is due to data acquisition in a local rather than an absolute LVR, highlighting the importance of using a rapid and zero pre-stress approach to characterise very soft and highly hydrated biological tissues.

Sphyga: a multiparameter open source tool for fabricating smart and tunable hydrogel microbeads.

Hydrogel microbeads are used in many biological applications, particularly for cell, protein or drug encapsulation. Although there are several methods for fabricating microbeads with controlled shapes and dimensions, many are limited to a small range of materials or sizes. We describe a compact open source tool-the spherical hydrogel generator (Sphyga)-for the fabrication of highly reproducible hydrogel based microbeads with predictable shapes and diameters ranging from 100 to 2000 µm. The unique feature of the system is the ability to modulate multiple parameters independently, so as to create a wide range of working conditions for fabricating tailored microbeads. Hence, by combining the different fabrication parameters, hydrogel beads with chosen shapes, sizes and materials can be generated with Sphyga. A multiparameter working-window was obtained by fixing the concentration of the base material, alginate, and varying the viscosity of the solution along with Sphyga's fabrication parameters (needle size, external air pressure, and material outflow). To validate the multiparameter working window, components such as proteins, cells, dyes and nanoparticles were also used to fabricate composite microbeads. The results show that the architecture of hydrogel microbeads can be engineered by considering the viscosity of the initial solution, which depends principally on the pH and composition of alginate solution. Coupled with Sphyga's multiple working parameters, material viscosity can then be used to tune hydrogel domains and thereby generate complex biologically relevant microenvironments for many biomedical applications.

Strain rate viscoelastic analysis of soft and highly hydrated biomaterials.

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 ( ɛ˙M 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 ɛ˙M, 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.

Mechanostructure and composition of highly reproducible decellularized liver matrices.

Despite the increasing number of papers on decellularized scaffolds, there is little consensus on the optimum method of decellularizing biological tissue such that the micro-architecture and protein content of the matrix are conserved as far as possible. Focusing on the liver, the aim of this study was therefore to develop a method for the production of well-characterized and reproducible matrices that best preserves the structure and composition of the native extra cellular matrix (ECM). Given the importance of matrix stiffness in regulating cell response, the mechanical properties of the decellularized tissue were also considered. The testing and analysis framework is based on the characterization of decellularized and untreated samples in the same reproducible initial state (i.e., the equilibrium swollen state). Decellularized ECM (dECM) were characterized using biochemical, histological, mechanical and structural analyses to identify the best procedure to ensure complete cell removal while preserving most of the native ECM structure and composition. Using this method, sterile decellularized porcine ECM with highly conserved intra-lobular micro-structure and protein content were obtained in a consistent and reproducible manner using the equilibrium swollen state of tissue or matrix as a reference. A significant reduction in the compressive elastic modulus was observed for liver dECM with respect to native tissue, suggesting a re-examination of design parameters for ECM-mimicking scaffolds for engineering tissues in vitro.

A phase diagram for microfabrication of geometrically controlled hydrogel scaffolds.

Hydrogels are considered as excellent candidates for tissue substitutes by virtue of their high water content and biphasic nature. However, the fact that they are soft, wet and floppy renders them difficult to process and use as custom-designed scaffolds. To address this problem alginate hydrogels were modeled and characterized by measuring stress-strain and creep behavior as well as viscosity as a function of sodium alginate concentration, cross-linking time and calcium ion concentration. The gels were then microfabricated into scaffolds using the pressure-assisted microsyringe. The mechanical and viscous characteristics were used to generate a processing window in the form of a phase diagram which describes the fidelity of the scaffolds as a function of the material and machine parameters. The approach can be applied to a variety of microfabrication methods and biomaterials in order to design well-controlled custom scaffolds.