Many nanocarrier cancer therapeutics currently under development, as well as those used in the clinical setting, rely upon the enhanced permeability and retention (EPR) effect to passively accumulate in the tumor microenvironment and kill cancer cells. In leukemia, where leukemogenic stem cells and their progeny circulate within the peripheral blood or bone marrow, the EPR effect may not be operative. Thus, for leukemia therapeutics, it is essential to target and bind individual circulating cells. Here, we investigate mesoporous silica nanoparticle (MSN)-supported lipid bilayers (protocells), an emerging class of nanocarriers, and establish the synthesis conditions and lipid bilayer composition needed to achieve highly monodisperse protocells that remain stable in complex media as assessed in vitro by dynamic light scattering and cryo-electron microscopy and ex ovo by direct imaging within a chick chorioallantoic membrane (CAM) model. We show that for vesicle fusion conditions where the lipid surface area exceeds the external surface area of the MSN and the ionic strength exceeds 20 mM, we form monosized protocells (polydispersity index <0.1) on MSN cores with varying size, shape, and pore size, whose conformal zwitterionic supported lipid bilayer confers excellent stability as judged by circulation in the CAM and minimal opsonization in vivo in a mouse model. Having established protocell formulations that are stable colloids, we further modified them with anti-EGFR antibodies as targeting agents and reverified their monodispersity and stability. Then, using intravital imaging in the CAM, we directly observed in real time the progression of selective targeting of individual leukemia cells (using the established REH leukemia cell line transduced with EGFR) and delivery of a model cargo. Overall, we have established the effectiveness of the protocell platform for individual cell targeting and delivery needed for leukemia and other disseminated disease.

Live tissues require vascular networks for cell nourishing. Mimicking the complex structure of native vascular networks in vitro requires understanding the governing factors of early tubulogenesis. Current vascularization protocols allow for spontaneous formation of vascular networks; however, there is still a need to provide control over the defined network structure. Moreover, there is little understanding on sprouting decision and migration, especially within 3D environments. Here, tessellated polymer scaffolds with various compartment geometries and a novel two‐step seeding protocol are used to study vessel sprouting decisions. Endothelial cells first organize into hollow vessels tracing the shape contour with high fidelity. Subsequent sprouts emerge in specific directions, responding to compartment geometry. Time‐lapse imaging is used to track vessel migration, evidencing that sprouts frequently emerge from the side centers, mainly migrating toward opposing corners, where the density of support cells (SCs) is the highest, providing the highest levels of angiogenic factors. SCs distribution is quantified by smooth muscle actin expression, confirming the cells preference for curved compartment surfaces and corners. Displacements within the hydrogel correlate with SCs distribution during the initial tubulogenesis phase. This work provides new insight regarding vessel sprouting decisions that should be considered when designing scaffolds for vascularized engineered tissues.

With the ever-increasing use of 3D cell models toward studying bio-nano interactions and offering alternatives to traditional 2D in vitro and in vivo experiments, methods to image biological tissue in real time and with high spatial resolution have become a must. A suitable technique therefore is surface-enhanced Raman scattering (SERS)-based microscopy, which additionally features reduced photocytotoxicity and improved light penetration. However, optimization of imaging and postprocessing parameters is still required. Herein we present a method to monitor cell proliferation over time in 3D, using multifunctional 3D-printed scaffolds composed of biologically inert poly(lactic-co-glycolic acid) (PLGA) as the base material, in which fluorescent labels and SERS-active gold nanoparticles (AuNPs) can be embedded. The combination of imaging techniques allows optimization of SERS imaging parameters for cell monitoring. The scaffolds provide anchoring points for cell adhesion, so that cell growth can be observed in a suspended 3D matrix, with multiple reference points for confocal fluorescence and SERS imaging. By prelabeling cells with SERS-encoded AuNPs and fluorophores, cell proliferation and migration can be simultaneously monitored through confocal Raman and fluorescence microscopy. These scaffolds provide a simple method to follow cell dynamics in 4D, with minimal disturbance to the tissue model.

The extracellular matrix (ECM) influences biological processes associated with tissue development and disease progression. However, robust cell‐free techniques to control fiber alignment of naturally derived ECM proteins, such as fibronectin (Fn), remain elusive. It is demonstrated that controlled hydrodynamics of Fn solutions at the air/fluid interface of porous tessellated polymer scaffolds (TPSs) generates suspended 3D fibrillar networks with alignment across multiple length scales (<1, 1–20 μm, extended to >1 mm). The direction of the fluid flow and the architecture of the polymeric supports influence protein solution flow profiles and, subsequently, the alignment of insoluble Fn fibrils. Aligned networks of fibrillar Fn characteristically alter fibroblast phenotype, indicated by increased directional orientation, enhanced nuclear and cytoskeletal polarity, and highly anisotropic and persistent cell motility when compared with nonaligned 3D networks and 2D substrates. Engineered extracellular matrices (EECMs) establish a critically needed tool for both fundamental and applied cell biology studies, with potential applications in diverse areas such as cancer biology and regenerative medicine.

Synthetic biological systems are used for a myriad of applications, including tissue engineered constructs for in vivo use and microengineered devices for in vitro testing. Recent advances in engineering complex biological systems have been fueled by opportunities arising from the combination of bioinspired materials with biological and computational tools. Driven by the availability of large datasets in the “omics” era of biology, the design of the next generation of tissue equivalents will have to integrate information from single‐cell behavior to whole organ architecture. Herein, recent trends in combining multiscale processes to enable the design of the next generation of biomaterials are discussed. Any successful microprocessing pipeline must be able to integrate hierarchical sets of information to capture key aspects of functional tissue equivalents. Micro‐ and biofabrication techniques that facilitate hierarchical control as well as emerging polymer candidates used in these technologies are also reviewed.

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