Biofunctionalized nanoparticles with pH-responsive and cell penetrating blocks for gene delivery.

Bridging the gap between nanoparticulate delivery systems and translational gene therapy is a long sought after requirement in nanomedicine-based applications. However, recent developments regarding nanoparticle functionalization have brought forward the ability to synthesize materials with biofunctional moieties that mimic the evolved features of viral particles. Herein we report the versatile conjugation of both cell penetrating arginine and pH-responsive histidine moieties into the chitosan polymeric backbone, to improve the physicochemical characteristics of the native material. Amino acid coupling was confirmed by 2D TOCSY NMR and Fourier transform infrared spectroscopy. The synthesized chitosan-histidine-arginine (CH-H-R) polymer complexed plasmid DNA biopharmaceuticals, and spontaneously assembled into stable 105 nm nanoparticles with spherical morphology and positive surface charge. The functionalized delivery systems were efficiently internalized into the intracellular compartment, and exhibited remarkably higher transfection efficiency than unmodified chitosan without causing any cytotoxic effect. Additional findings regarding intracellular trafficking events reveal their preferential escape from degradative lysosomal pathways and nuclear localization. Overall, this assembly of nanocarriers with bioinspired moieties provides the foundations for the design of efficient and customizable materials for cancer gene therapy.

Formulation of chitosan-TPP-pDNA nanocapsules for gene therapy applications.

The encapsulation of DNA inside nanoparticles meant for gene delivery applications is a challenging process where several parameters need to be modulated in order to design nanocapsules with specific tailored characteristics. The purpose of this study was to investigate and improve the formulation parameters of plasmid DNA (pDNA) loaded in chitosan nanocapsules using tripolyphosphate (TPP) as polyanionic crosslinker. Nanocapsule morphology and encapsulation efficiency were analyzed as a function of chitosan degree of deacetylation and chitosan-TPP ratio. The manipulation of these parameters influenced not only the particle size but also the encapsulation and release of pDNA. Consequently the transfection efficiency of the nanoparticulated systems was also enhanced with the optimization of the particle characteristics. Overall, the differently formulated nanoparticulated systems possess singular properties that can be employed according to the desired gene delivery application.

Bioinstructive microparticles for self-assembly of mesenchymal stem Cell-3D tumor spheroids.

3D multicellular tumor spheroids (3D-MCTS) that closely mimic in vitro the complex lung tumor microenvironment (TME) are highly desirable for screening innovative anti-cancer therapeutics. Despite significant improvements in mimicking lung TME, few models have combined tumor-infiltrating mesenchymal stem cells from bone marrow (hBM-MSCs) with heterotypic 3D tumor spheroid models containing ECM mimetic components. Herein, we engineered hybrid 3D-MCTS that combine, for the first time, A549:fibroblasts:hBM-MSCs in heterotypic tri-culture, with bioinstructive hyaluronan microparticles that act as tumor-ECM mimetics and as cell-anchoring hotspots. The obtained results indicated that 3D microspheres provided proper support for cells to self-assemble into compact 3D microtissues and promoted an increase in CD44 expression, emulating the presence of native-ECM hyaluronan. 3D-MCTS size and sphere-like morphology was reproducible and tri-culture models presented the characteristic solid tumors necrotic core. Mesenchymal stem cells tracking demonstrated that hBM-MSCs migrate to different regions in 3D microtumors mass exhibiting dynamic interactions with cancer cells and stromal fibroblasts, alike in human tumors. Importantly, doxorubicin administration revealed hBM-MSCs effect on cytotoxic responses in 3D tri-culture models and in dual cultures of hBM-MSCs:A549 at 10:1 ratio. Such findings evidence the relevance of including hBM-MSCs in combination with cancer-stromal fibroblasts in 3D in vitro tumor models and the importance to test different cell-to-cell ratios to mimic tumor heterogeneity. In addition, bioinstructive hyaluronan-microparticles were also effective as cell-agglomerating scaffolds and showed potential to be used as an enabling technology for including different ECM components in 3D in vitro models in the future.

Design of spherically structured 3D in vitro tumor models -Advances and prospects.

Three-dimensional multicellular tumor models are receiving an ever-growing focus as preclinical drug-screening platforms due to their potential to recapitulate major physiological features of human tumors in vitro. In line with this momentum, the technologies for assembly of 3D microtumors are rapidly evolving towards a comprehensive inclusion of tumor microenvironment elements. Customized spherically structured platforms, including microparticles and microcapsules, provide a robust and scalable technology to imprint unique biomolecular tumor microenvironment hallmarks into 3D in vitro models. Herein, a comprehensive overview of novel advances on the integration of tumor-ECM components and biomechanical cues into 3D in vitro models assembled in spherical shaped platforms is provided. Future improvements regarding spatiotemporal/mechanical adaptability, and degradability, during microtumors in vitro 3D culture are also critically discussed considering the realistic potential of these platforms to mimic the dynamic tumor microenvironment. From a global perspective, the production of 3D multicellular spheroids with tumor ECM components included in spherical models will unlock their potential to be used in high-throughput screening of therapeutic compounds. It is envisioned, in a near future, that a combination of spherically structured 3D microtumor models with other advanced microfluidic technologies will properly recapitulate the flow dynamics of human tumors in vitro. STATEMENT OF SIGNIFICANCE: The ability to correctly mimic the complexity of the tumor microenvironment in vitro is a key aspect for the development of evermore realistic in vitro models for drug-screening and fundamental cancer biology studies. In this regard, conventional spheroid-based 3D tumor models, combined with spherically structured biomaterials, opens the opportunity to precisely recapitulate complex cell-extracellular matrix interactions and tumor compartmentalization. This review provides an in-depth focus on current developments regarding spherically structured scaffolds engineered into in vitro 3D tumor models, and discusses future advances toward all-encompassing platforms that may provide an improved in vitro/in vivo correlation in a foreseeable future.

Chitosan/arginine-chitosan polymer blends for assembly of nanofibrous membranes for wound regeneration.

Frequently, skin is subjected to damaging events, such as deep cuts, burns or ulcers, which may compromise the integrity of this organ. To overcome such lesions, different strategies have been employed. Among them, wound dressings aimed to re-establish skin native properties and decreased patient pain have been pursued for a long time. Herein, an electrospun membrane comprised by deacetylated/arginine modified chitosan (CH-A) was produced to be used as a wound dressing. The obtained results showed that the membrane has a highly hydrophilic and porous three-dimensional nanofibrous network similar to that found in human native extracellular matrix. In vitro data indicate that human fibroblasts adhere and proliferate in contact with membranes, thus corroborating their biocompatibility. This nanofiber-based biomaterial also demonstrated bactericidal activity for two bacterial strains. In vivo application of CH-A nanofibers in full thickness wounds resulted in an improved tissue regeneration and faster wound closure, when compared to non-modified membranes. Such findings support the suitability of using this membrane as a wound dressing in a near future.

Manufacture of ß-TCP/alginate scaffolds through a Fab@home model for application in bone tissue engineering.

The growing need to treat bone-related diseases in an elderly population compels the development of novel bone substitutes to improve patient quality of life. In this context, the advent of affordable and effective rapid prototyping equipment, such as the Fab@home plotter, has contributed to the development of novel scaffolds for bone tissue engineering. In this study, we report for the first time the use of a Fab@home plotter for the production of 3D scaffolds composed by beta-tricalcium phosphate (ß-TCP)/alginate hybrid materials. ß-TCP/alginate mixtures were used in a proportion of 50/50% (w/w), 30/70% (w/w) and 20/80% (w/w). The printing parameters were optimized to a nozzle diameter of 20 Gauge for the production of rigid scaffolds with pre-defined architectures. We observed that, despite using similar printing parameters, both the precision and resolution of the scaffolds were significantly affected by the blend's viscosity. In particular, we demonstrate that the higher viscosity of 50/50 scaffolds (150.0 ± 3.91 mPa s) provides a higher precision in the extrusion process. The physicochemical and biological characterization of the samples demonstrated that the 50/50 scaffolds possessed a resistance to compression comparable to that of native trabecular bone. Moreover, this particular formulation also exhibited a Young's modulus that was higher than that of trabecular bone. Scanning electron microscopy and fluorescence microscopy analysis revealed that osteoblasts were able to adhere, proliferate and also penetrate into the scaffold's architecture. Altogether, our findings suggest that the Fab@home printer can be employed in the manufacture of reproducible scaffolds, using a formulation 50/50 alginate-ß-TCP that has suitable properties to be applied as bone substitutes in the future.

Bioactive polymeric-ceramic hybrid 3D scaffold for application in bone tissue regeneration.

The regeneration of large bone defects remains a challenging scenario from a therapeutic point of view. In fact, the currently available bone substitutes are often limited by poor tissue integration and severe host inflammatory responses, which eventually lead to surgical removal. In an attempt to address these issues, herein we evaluated the importance of alginate incorporation in the production of improved and tunable ß-tricalcium phosphate (ß-TCP) and hydroxyapatite (HA) three-dimensional (3D) porous scaffolds to be used as temporary templates for bone regeneration. Different bioceramic combinations were tested in order to investigate optimal scaffold architectures. Additionally, 3D ß-TCP/HA vacuum-coated with alginate, presented improved compressive strength, fracture toughness and Young's modulus, to values similar to those of native bone. The hybrid 3D polymeric-bioceramic scaffolds also supported osteoblast adhesion, maturation and proliferation, as demonstrated by fluorescence microscopy. To the best of our knowledge this is the first time that a 3D scaffold produced with this combination of biomaterials is described. Altogether, our results emphasize that this hybrid scaffold presents promising characteristics for its future application in bone regeneration.