Electrospun nanofibers for 3-D cancer models, diagnostics, and therapy.

As one of the leading causes of global mortality, cancer has prompted extensive research and development to advance efficacious drug discovery, sustained drug delivery and improved sensitivity in diagnosis. Towards these applications, nanofibers synthesized by electrospinning have exhibited great clinical potential as a biomimetic tumor microenvironment model for drug screening, a controllable platform for localized, prolonged drug release for cancer therapy, and a highly sensitive cancer diagnostic tool for capture and isolation of circulating tumor cells in the bloodstream and for detection of cancer-associated biomarkers. This review provides an overview of applied nanofiber design with focus on versatile electrospinning fabrication techniques. The influence of topographical, physical, and biochemical properties on the function of nanofiber assemblies is discussed, as well as current and foreseeable barriers to the clinical translation of applied nanofibers in the field of oncology.

Chitosan-based composite bilayer scaffold as an in vitro osteochondral defect regeneration model.

Prolonged osteochondral tissue damage can result in osteoarthritis and decreased quality of life. Multiphasic scaffolds, where different layers model different microenvironments, are a promising treatment approach, yet stable joining between layers during fabrication remains challenging. Here, a bilayer scaffold for osteochondral tissue regeneration was fabricated using thermally-induced phase separation (TIPS). Two distinct polymer solutions were layered before TIPS, and the resulting porous, bilayer scaffold was characterized by seamless interfacial integration and a mechanical stiffness gradient reflecting the native osteochondral microenvironment. Chitosan is a critical component of both scaffold layers to facilitate cell attachment and the formation of polyelectrolyte complexes with other biologically relevant natural polymers. The articular cartilage region was optimized for hyaluronic acid content and stiffness, while the subchondral bone region was defined by higher stiffness and osteoconductive hydroxyapatite content. Following co-culture with chondrocyte-like (SW-1353 or mesenchymal stem cells) and osteoblast-like cells (MG63), cell proliferation and migration to the interface along with increased gene expression associated with relevant markers of osteogenesis and chondrogenesis indicates the potential of this bilayer scaffold for osteochondral tissue regeneration.

Hyaluronic Acid-Coated Aligned Nanofibers for the Promotion of Glioblastoma Migration.

The propensity of glioblastoma multiforme (GBM) cells to migrate along white matter tracts and blood vessels suggests that topographical cues associated with brain parenchyma greatly influence GBM motility and invasion. In vitro cell culture platforms that mimic the physical and biochemical characteristics of brain tissue are needed to develop biologically relevant GBM migration models for the development of anticancer therapies. Here, we fabricated highly aligned chitosan-polycaprolactone (C-PCL) polyblend nanofibers coated with hyaluronic acid (HA), a glycosaminoglycan commonly found in the brain, to simulate the structure and biochemistry of native brain tissue. The influence of topography on GBM cell behavior was apparent on both HA-coated and uncoated nanofibers where cells aligned axially along nanofibers and displayed an elongated morphology associated with migration. Time lapse imaging revealed that migrating cells on nanofibers were less likely to divide, suggesting a shift to a mesenchymal-like phenotype. Cells cultured on nanofibers coated with 0.5% HA achieved the highest migratory speed relative to uncoated nanofibers and 2D adherent cultures on polystyrene plates. Further, cells on nanofibers were more resistant to cell death, after exposure to the common chemotherapeutic Temozolomide than cells grown on 2D polystyrene plates. These results indicate that HA-coated nanofibers are a promising substrate for characterization of GBM migration and investigation of novel therapies.

Fabrication and Characterization of Chitosan-Hyaluronic Acid Scaffolds with Varying Stiffness for Glioblastoma Cell Culture.

The invasive and recurrent nature of glioblastoma multiforme (GBM) is linked to a small subpopulation of cancer cells, which are self-renewing, resistant to standard treatment regimens, and induce formation of new tumors. Matrix stiffness is implicated in the regulation of cell proliferation, drug resistance, and reversion to a more invasive phenotype. Therefore, understanding the relationship between matrix stiffness and tumor cell behavior is vital to develop appropriate in vitro tumor models. Here, chitosan-hyaluronic acid (CHA) polyelectrolyte complex scaffolds are fabricated with statistically significant stiffness variances to characterize the effect of scaffold stiffness on morphology, proliferation, drug resistance, and gene expression in human glioblastoma cells (U-87 MG). All scaffolds support GBM proliferation over a 12-day culture period, yet larger spheroids are observed in scaffolds with higher stiffness. Additionally, GBM cells cultured in stiffer CHA scaffolds prove significantly more resistant to the common chemotherapeutic temozolomide. Moreover, the stiffer 8% CHA scaffolds exhibit an increase in expression of drug resistance and invasion related genes compared to 2D culture. CHA scaffolds present a tunable microenvironment for enhanced tumor cell malignancy and may provide a valuable in vitro microenvironment for studying tumor progression and screening anticancer therapies.

Chitosan-Poly(caprolactone) Nanofibers for Skin Repair.

Dermal wounds, both acute and chronic, represent a significant clinical challenge and therefore the development of novel biomaterial-based skin substitutes to promote skin repair is essential. Nanofibers have garnered attention as materials to promote skin regeneration due to the similarities in morphology and dimensionality between nanofibers and native extracellular matrix proteins, which are critical in guiding cutaneous wound healing. Electrospun chitosan-poly(caprolactone) (CPCL) nanofiber scaffolds, which combine the important intrinsic biological properties of chitosan and the mechanical integrity and stability of PCL, were evaluated as skin tissue engineering scaffolds using a mouse cutaneous excisional skin defect model. Gross assessment of wound size and measurement of defect recovery over time as well as histological evaluation of wound healing showed that CPCL nanofiber scaffolds increased wound healing rate and promoted more complete wound closure as compared with Tegaderm, a commercially available occlusive dressing. CPCL nanofiber scaffolds represent a biomimetic approach to skin repair by serving as an immediately available provisional matrix to promote wound closure. These nanofiber scaffolds may have significant potential as a skin substitute or as the basis for more complex skin tissue engineering constructs involving integration with biologics.

Culture on 3D Chitosan-Hyaluronic Acid Scaffolds Enhances Stem Cell Marker Expression and Drug Resistance in Human Glioblastoma Cancer Stem Cells.

The lack of in vitro models that support the growth of glioblastoma (GBM) stem cells (GSCs) that underlie clinical aggressiveness hinders developing new, effective therapies for GBM. While orthotopic patient-derived xenograft models of GBM best reflect in vivo tumor behavior, establishing xenografts is a time consuming, costly, and frequently unsuccessful endeavor. To address these limitations, a 3D porous scaffold composed of chitosan and hyaluronic acid (CHA) is synthesized. Growth and expression of the cancer stem cell (CSC) phenotype of the GSC GBM6 taken directly from fresh xenogratfs grown on scaffolds or as adherent monolayers is compared. While 2D adherent cultures grow as monolayers of flat epitheliod cells, GBM6 cells proliferate within pores of CHA scaffolds as clusters of self-adherent ovoid cells. Growth on scaffolds is accompanied by greater expression of genes that mediate epithelial-mesenchymal transition and maintain a primitive, undifferentiated phenotype, hallmarks of CSCs. Scaffold-grown cells also display higher expression of genes that promote resistance to hypoxia-induced oxidative stress. In accord, scaffold-grown cells show markedly greater resistance to clinically utilized alkylating agents compared to adherent cells. These findings suggest that our CHA scaffolds better mimic in vivo biological and clinical behavior and provide insights for developing novel individualized treatments.

3D Porous Chitosan-Alginate Scaffolds Promote Proliferation and Enrichment of Cancer Stem-Like Cells.

Cancer stem cells are increasingly becoming a primary target for new cancer treatment development. The ability to study their transient behavior in vitro will provide the opportunity for high-throughput testing of more effective therapies. We have previously demonstrated the use of 3D porous chitosan-alginate (CA) scaffolds to promote cancer stem-like cell (CSC) proliferation and enrichment in glioblastoma. Here we use 3D porous CA scaffolds to promote cancer stem-like cell enrichment in cell lines from prostate, liver, and breast cancers, and investigate the proliferation, morphology, and gene expressions of cells cultured in CA scaffolds as compared to 2D controls. The 3D CA scaffold cultures for all three cancer types showed reduced proliferation, formation of tumor spheroids, and increased expression of CSC associated mark genes (CD133 and NANOG), as opposed to monolayers. Additionally, we present a putative mechanism for the cancer stem-like cell enrichment on CA scaffolds. This study demonstrates that the cancer stem-like cell enrichment in CA scaffolds is a robust process that is not restricted to particular cancer types.

Modeling the tumor microenvironment using chitosan-alginate scaffolds to control the stem-like state of glioblastoma cells.

Better prediction of in vivo drug efficacy using in vitro models should greatly improve in vivo success. Here we utilize 3D highly porous chitosan-alginate complex scaffolds to probe how various components of the glioblastoma microenvironment including extracellular matrix and stromal cells affect tumor cell stem-like state.

High-throughput and high-yield fabrication of uniaxially-aligned chitosan-based nanofibers by centrifugal electrospinning.

The inability to produce large quantities of nanofibers has been a primary obstacle in advancement and commercialization of electrospinning technologies, especially when aligned nanofibers are desired. Here, we present a high-throughput centrifugal electrospinning (HTP-CES) system capable of producing a large number of highly-aligned nanofiber samples with high-yield and tunable diameters. The versatility of the design was revealed when bead-less nanofibers were produced from copolymer chitosan/polycaprolactone (C-PCL) solutions despite variations in polymer blend composition or spinneret needle gauge. Compared to conventional electrospinning techniques, fibers spun with the HTP-CES not only exhibited superior alignment, but also better diameter uniformity. Nanofiber alignment was quantified using Fast Fourier Transform (FFT) analysis. In addition, a concave correlation between the needle diameter and resultant fiber diameter was identified. This system can be easily scaled up for industrial production of highly-aligned nanofibers with tunable diameters that can potentially meet the requirements for various engineering and biomedical applications.

Thermoreversible poly(ethylene glycol)-g-chitosan hydrogel as a therapeutic T lymphocyte depot for localized glioblastoma immunotherapy.

The outcome for glioblastoma patients remains dismal for its invariably recrudesces within 2 cm of the resection cavity. Local immunotherapy has the potential to eradicate the residual infiltrative component of these tumors. Here, we report the development of a biodegradable hydrogel containing therapeutic T lymphocytes for localized delivery to glioblastoma cells for brain tumor immunotherapy. Thermoreversible poly(ethylene glycol)-g-chitosan hydrogels (PCgels) were optimized for steady T lymphocyte release. Nuclear magnetic resonance spectroscopy confirmed the chemical structure of poly(ethylene glycol)-g-chitosan, and rheological studies revealed that the sol-to-gel transition of the PCgel occurred around ≥32 °C. T lymphocyte invasion through the PCgel and subsequent cytotoxicity to glioblastoma were assessed in vitro. The PCgel was shown to be cellular compatible with T lymphocytes, and the T lymphocytes retain their anti-glioblastoma activity after being encapsulated in the PCgel. T lymphocytes in the PCgel were shown to be more effective in killing glioblastoma than those in the Matrigel control. This may be attributed to the optimal pore size of the PCgel allowing better invasion of T lymphocytes. Our study suggests that this unique PCgel depot may offer a viable approach for localized immunotherapy for glioblastoma.

Chitosan-based thermoreversible hydrogel as an in vitro tumor microenvironment for testing breast cancer therapies.

Breast cancer is a major health problem for women worldwide. Although in vitro culture of established breast cancer cell lines is the most widely used model for preclinical assessment, it poorly represents the behavior of breast cancers in vivo. Acceleration of the development of effective therapeutic strategies requires a cost-efficient in vitro model that can more accurately resemble the in vivo tumor microenvironment. Here, we report the use of a thermoreversible poly(ethylene glycol)-g-chitosan hydrogel (PCgel) as an in vitro breast cancer model. We hypothesized that PCgel could provide a tumor microenvironment that promotes cultured cancer cells to a more malignant phenotype with drug and immune resistance. Traditional tissue culture plates and Matrigel were applied as controls in our studies. In vitro cellular proliferation and morphology, the secretion of angiogenesis-related growth factors and cytokines, and drug and immune resistance were assessed. Our results show that PCgel cultures promoted tumor aggregate formation, increased secretion of various angiogenesis- and metastasis-related growth factors and cytokines, and increased tumor cell resistance to chemotherapeutic drugs and immunotherapeutic T cells. This PCgel platform may offer a valuable strategy to bridge the gap between standard in vitro and costly animal studies for a wide variety of experimental designs.