Developing nanostructured thin films as biomimetic tissue-engineered platform for cancer research

Engineering in vitro models that reproduce tumor microenvironment and mimic functions and responses of tissues that is more physiologically relevant represents a potential bridge to cover the gap between animal models and clinical studies. In this talk, we describe nanostructured thin films as templates to develop biomimetic tissue-engineered technologies for cancer research. Our model systems enables us to examine the impact of dynamics changes in the physical environment of tumor microenvironment (TME) in conjunction to tumor-stromal (fibroblasts, mesenchymal stem cells (MSCs), immune cells) cell interactions to potentially mimic stable disease and/or its eventual progression to advanced stages. Tumors actively modulate their microenvironment by recruiting MSCs, lymphocytes and macrophages; vascular endothelial cells; and tumor-associated stromal cells such as fibroblasts. Tumor progression results in dynamic changes in the cell-cell interaction and tumor biology. Currently, the impact of key tumor-stromal cell interactions is unknown due to the lack of models or approaches that can address this key question. In this study, we report a robust, inexpensive, protein free method that utilizes polyelectrolyte multilayers (PEMs) and capillary force lithography (CFL) to generate patterned co-culture models of breast cancer cells and stromal cells. PEMs have been shown to be excellent candidates for biomaterial applications. In our study, we used synthetic polymers, namely poly(diallyldimethylammoniumchloride) (PDAC) and sulfonated poly(styrene) (SPS) as the polycation and polyanion, respectively, to build the multilayers. We as well others have previously shown that PEM surfaces utilizing PDAC and SPS also provide an ability to control the arrangement of multiple cell types with subcellular resolution. This technique allows the formation of cell patterns with different shapes and sizes of tunable directional properties, recreating cell-cell interactions in a highly controlled manner. In this study, we capitalized upon the differential cell attachment and spreading of breast cancer cells on different PEM surfaces to engineer patterned co-cultures of breast cancer cells and stromal cells. To demonstrate the translational validity of our platform, we employed two developmentally distinct human breast cell lines for co-culture development: 1) BT474 (HER2+ invasive breast cancer cells to model invasive ductal carcinoma (IDC)), and 2) 21MT-1 (stable patient-derived metastatic breast cancer cells isolated from the metastatic pleural effusion to model invasive mammary carcinoma (IMC)). We also used two different types of stromal cells, mammary epithelial cells (MCF10A) and mesenchymal stem cells (MSCs) to demonstrate the versatility of our platform. Since MCF10A are non-tumorigenic cells and MSCs have a significant role in metastasis, our platform provides an opportunity to study cell-cell interactions in a heterogeneous TME, an inimitable property of cancer progression. We further illustrated that our in vitro breast tumor model is capable of staging the breast tumor dynamics and emulating clinical relevant molecular pathways at different stages of tumor points. For this purpose, we utilized the co-culture system developed in this study and demonstrated that our platform simulated key clinical markers prominently used for tumor diagnosis, including tumor (HER-2) and proliferation (Ki67) markers. Also our platform mirrored the clinical conditions when probed for miRNA-21 and miRNA-34 expression. The development of such in vitro models that recapitulates the in vivo like signaling in tumor would be desirable to increase the drive towards precision medicine to identify key biomarkers for early diagnosis and novel therapeutic interventions

SUBSTRATE DELIVERY OF EMBEDDED LIPOSOMES

This invention relates to compositions useful for localized and sustained release of therapeutic agents, and more particularly to functionalized liposomes embedded in a poly electrolyte multilayer. Methods of preparing the compositions, methods of treating diseases, devices, and pharmaceutical compositions comprising the compositions are also provided

SUBSTRATE DELIVERY OF EMBEDDED LIPOSOMES

This invention relates to compositions useful for localized and sustained release of therapeutic agents , and more par ticularly to functionalized liposomes embedded in a poly electrolyte multilayer . Methods of preparing the composi tions , methods of treating diseases , devices , and pharmaceutical compositions comprising the compositions are also provide

Tunable Resistive m-dPEG Acid Patterns on Polyelectrolyte Multilayers at Physiological Conditions: Template for Directed Deposition of Biomacromolecules

This paper describes a new class of salt-responsive poly(ethylene glycol) (PEG) self-assembled monolayers (SAMs) on top of polyelectrolyte multilayer (PEMs) films. PEM surfaces with poly(diallyldimethylammonium chloride) as the topmost layer are chemically patterned by microcontact printing (μCP) oligomeric PEG molecules with an activated carboxylic acid terminal group (m-dPEG acid). The resistive m-d-poly(ethylene glycol) (m-dPEG) acid molecules on the PEMs films were subsequently removed from the PEM surface with salt treatment, thus converting the nonadhesive surfaces into adhesive surfaces. The resistive PEG patterns facilitate the directed deposition of various macromolecules such as polymers, dyes, colloidal particles, proteins, liposomes, and nucleic acids. Further, these PEG patterns act as a universal resist for different types of cells (e.g., primary cells, cell lines), thus permitting more flexibility in attaching a wide variety of cells to material surfaces. The patterned films were characterized by optical microscopy and atomic force microscopy (AFM). The PEG patterns were removed from the PEM surface at certain salt conditions without affecting the PEM films underneath the SAMs. Removal of the PEG SAMs and the stability of the PEM films underneath it were characterized with ellipsometry and optical microscopy. Such salt- and pH-responsive surfaces could lead to significant advances in the fields of tissue engineering, targeted drug delivery, materials science, and biology

SECs (Sinusoidal Endothelial Cells), Liver Microenvironment, and Fibrosis

Liver fibrosis is awound-healing response to chronic liver injury such as alcoholic/nonalcoholic fatty liver disease and viral hepatitis with no FDA-approved treatments. Liver fibrosis results in a continual accumulation of extracellular matrix (ECM) proteins and paves the way for replacement of parenchyma with nonfunctional scar tissue. The fibrotic condition results in drastic changes in the local mechanical, chemical, and biological microenvironment of the tissue. Liver parenchyma is supported by an efficient network of vasculature lined by liver sinusoidal endothelial cells (LSECs). These nonparenchymal cells are highly specialized resident endothelial cell type with characteristic morphological and functional features. Alterations in LSECs phenotype including lack of LSEC fenestration, capillarization, and formation of an organized basement membrane have been shown to precede fibrosis and promote hepatic stellate cell activation. Here, we review the interplay of LSECs with the dynamic changes in the fibrotic liver microenvironment such as matrix rigidity, altered ECM protein profile, and cell-cell interactions to provide insight into the pivotal changes in LSEC physiology and the extent towhich it mediates the progression of liver fibrosis. Establishing the molecular aspects of LSECs in the light of fibrotic microenvironment is valuable towards development of novel therapeutic and diagnostic targets of liver fibrosis

Protocol to engineer nanofilms embedded lipid nanoparticles for controlled and targeted drug delivery (NECTAR)

We present a protocol to engineer a substrate-mediated delivery platform comprising hyaluronic acid-coated lipid nanoparticles (HALNPs) embedded into polyelectrolyte multilayer (PEM) films. This platform allows controlled spatiotemporal release of lipid nanoparticles (LNP) by embedding them within the polyelectrolyte multilayer films matrix. HALNP conjugate with antibodies also adds the ability for targeted delivery. The use of LNP enables this platform to encapsulate both hydrophobic and hydrophilic drugs. This platform can easily be reproduced and utilized for various biomedical drug delivery applications. For complete details on the use and execution of this protocol, please refer to Hayward et al. (2015, 2016a, 2016b), Hayward and Kidambi (2018), and Kidambi and Hayward (2022)

High Expression of Glycolytic Genes in Clinical Glioblastoma Patients Correlates With Lower Survival

Glioblastoma (GBM), the most aggressive brain tumor, is associated with a median survival at diagnosis of 16–20 months and limited treatment options. The key hallmark of GBM is altered tumor metabolism and marked increase in the rate of glycolysis. Aerobic glycolysis along with elevated glucose consumption and lactate production supports rapid cell proliferation and GBM growth. In this study, we examined the gene expression profile of metabolic targets in GBM samples from patients with lower grade glioma (LGG) and GBM. We found that gene expression of glycolytic enzymes is up-regulated in GBM samples and significantly associated with an elevated risk for developing GBM. Our findings of clinical outcomes showed that GBM patients with high expression of HK2 and PKM2 in the glycolysis related genes and low expression of genes involved in mitochondrial metabolism-SDHB and COX5A related to tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS), respectively, was associated with poor patient overall survival. Surprisingly, expression levels of genes involved in mitochondrial oxidative metabolism are markedly increased in GBM compared to LGG but was lower compared to normal brain. The fact that in GBM the expression levels of TCA cycle and OXPHOS-related genes are higher than those in LGG patients suggests the metabolic shift in GBM cells when progressing from LGG to GBM. These results are an important step forward in our understanding of the role of metabolic reprogramming in glioma as drivers of the tumor and could be potential prognostic targets in GBM therapies

Protocol to engineer nanofilms embedded lipid nanoparticles for controlled and targeted drug delivery (NECTAR)

We present a protocol to engineer a substrate-mediated delivery platform comprising hyaluronic acid-coated lipid nanoparticles (HALNPs) embedded into polyelectrolyte multilayer (PEM) films. This platform allows controlled spatiotemporal release of lipid nanoparticles (LNP) by embedding them within the polyelectrolyte multilayer films matrix. HALNP conjugate with antibodies also adds the ability for targeted delivery. The use of LNP enables this platform to encapsulate both hydrophobic and hydrophilic drugs. This platform can easily be reproduced and utilized for various biomedical drug delivery applications. For complete details on the use and execution of this protocol, please refer to Hayward et al. (2015, 2016a, 2016b), Hayward and Kidambi (2018), and Kidambi and Hayward (2022)

Hyaluronic acid-conjugated liposome nanoparticles for targeted delivery to CD44 overexpressing glioblastoma cells

Glioblastoma Multiforme (GBM) is a highly prevalent and deadly brain malignancy characterized by poor prognosis and restricted disease management potential. Despite the success of nanocarrier systems to improve drug/gene therapy for cancer, active targeting specificity remains a major hurdle for GBM. Additionally, since the brain is a multi-cell type organ, there is a critical need to develop an approach to distinguish between GBM cells and healthy brain cells for safe and successful treatment. In this report, we have incorporated hyaluronic acid (HA) as an active targeting ligand for GBM. To do so, we employed HA conjugated liposomes (HALNPs) to study the uptake pathway in key cells in the brain including primary astrocytes, microglia, and human GBM cells. We observed that the HALNPs specifically target GBM cells over other brain cells due to higher expression of CD44 in tumor cells. Furthermore, CD44 driven HALNP uptake into GBM cells resulted in lysosomal evasion and increased efficacy of Doxorubicin, a model anti-neoplastic agent, while the astrocytes and microglia cells exhibited extensive HALNP-lysosome co-localization and decreased antineoplastic potency. In summary, novel CD44 targeted lipid based nanocarriers appear to be proficient in mediating site-specific delivery of drugs via CD44 receptors in GBM cells, with an improved therapeutic margin and safety

Astrogliosis in a dish: Substrate stiffness induces astrogliosis in primary rat astrocytes

Astrogliosis due to brain injury or disease can lead to varying molecular and morphological changes in astrocytes. Magnetic resonance elastography and ultrasound have demonstrated that brain stiffness varies with age and disease state. However, there is a lack in understanding the role of varied stiffness on the progression of astrogliosis highlighting a critical need to engineer in vitro models that mimic disease stages. Such models need to incorporate the dynamic changes in the brain microenvironment including the stiffness changes. In this study we developed a polydimethyl siloxane (PDMS) based platform that modeled the physiologically relevant stiffness of brain in both a healthy (200 Pa) and diseased (8000 Pa) state to investigate the effect of stiffness on astrocyte function. We observed that astrocytes grown on soft substrates displayed a consistently more quiescent phenotype while those on stiff substrates displayed an astrogliosis-like morphology. In addition to morphological changes, astrocytes cultured on stiff substrates demonstrated significant increase in other astrogliosis hallmarks – cellular proliferation and glial fibrillary acidic protein (GFAP) protein expression. Furthermore, culturing astrocytes on a stiff surface resulted in increased reactive oxygen species (ROS) production, increased super oxide dismutase activity and decreased glutamate uptake. Our platform lends itself for study of potential therapeutic strategies for brain injury focusing on the intricate brain microenvironment-astrocytes signaling pathways