Multifunctional albumin nanoparticles as combination drug carriers for intra-tumoral chemotherapy.

Current cancer therapies are challenged by weakly soluble drugs and by drug combinations that exhibit non-uniform biodistribution and poor bioavailability. In this study, we have presented a new platform of advanced healthcare materials based on albumin nanoparticles (ANPs) engineered as tumor penetrating, delivery vehicles of combinatorially applied factors to solid tumors. These materials were designed to overcome three sequential key barriers: tissue level transport across solid tumor matrix; uptake kinetics into individual cancer cells; therapeutic resistance to single chemotherapeutic drugs. The ANPs were designed to penetrate deeper into solid tumor matrices using collagenase decoration and evaluated using a three-dimensional multicellular melanoma tumor spheroid model. Collagenase modified ANPs exhibited 1-2 orders of magnitude greater tumor penetration than unmodified ANPs into the spheroid mass after 96 hours, and showed preferential uptake into individual cancer cells for smaller sized ANPs (<100 nm). For enhanced efficacy, collagenase coated ANPs were modified with two therapeutic agents, curcumin and riluzole, with complementary mechanisms of action for combined cell cycle arrest and apoptosis in melanoma. The collagenase coated, drug loaded nanoparticles induced significantly more cell death within 3-D tumor models than the unmodified, dual drug loaded ANP particles and the kinetics of cytotoxicity was further influenced by the ANP size. Thus, multifunctional nanoparticles can be imbued with complementary size and protease activity features that allow them to penetrate solid tumors and deliver combinatorial therapeutic payload with enhanced cancer cytotoxicity but minimal collateral damage to healthy primary cells.

The effect of hypoxia on in vitro prevascularization of a thick soft tissue.

Prevascularizing an implantable tissue is one strategy to improve oxygen (O(2)) transport throughout larger tissues upon implantation. This study examined the role of hypoxia both during (i.e., as a stimulus) and after (i.e., mimicking implant conditions) vascularization of an implantable tissue. Tissues consisted of microcarrier beads coated with human umbilical vein endothelial cells embedded in fibrin. The fibrin was covered with a monolayer of normal human lung fibroblasts (NHLFs), or exposed to conditioned media from NHLFs. Capillary networks developed at 20% or 1% O(2) tension for 8 days. In some experiments, tissues were supplemented with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor, whereas in others the tissues prevascularized at 20% O(2) were transferred to 1% O(2) for 8 additional days. Maximal capillary formation occurred in media conditioned by NHLFs at 20% O(2), supplemented with VEGF (concentration >10 pM). Hypoxia (1% O(2)) did not stimulate basic fibroblast growth factor production and decreased in vitro angiogenesis, despite an increase in endogenous VEGF production. Hypoxia also degraded a preformed capillary network within 4 days. Hence, strategies to prevascularize implantable tissues may not require the physical presence of stromal cells, but will likely require fibroblast-derived growth factors in addition to VEGF to maintain capillary growth.

Prevascularization of a fibrin-based tissue construct accelerates the formation of functional anastomosis with host vasculature.

One critical obstacle facing tissue engineering is the formation of functional vascular networks that can support tissue survival in vivo. We hypothesized that prevascularizing a tissue construct with networks of well-formed capillaries would accelerate functional anastomosis with the host upon implantation. Fibrin-based tissues were prevascularized with capillary networks by coculturing human umbilical vein endothelial cells (HUVECs) and fibroblasts in fibrin gels for 1 week. The prevascularized tissue and nonprevascularized controls were implanted subcutaneously onto the dorsal surface of immune-deficient mice and retrieved at days 3, 5, 7 and 14. HUVEC-lined vessels containing red blood cells were evident in the prevascularized tissue by day 5, significantly earlier than nonprevascularized tissues (14 days). Analysis of the HUVEC-lined vessels demonstrated that the number and area of perfused lumens in the prevascularized tissue were significantly larger compared to controls. In addition, collagen deposition and a larger number of proliferating cells were evident in the prevascularized tissue at day 14. Our results demonstrate that prevascularizing a fibrin-based tissue with well-formed capillaries accelerates anastomosis with the host vasculature, and promotes cellular activity consistent with tissue remodeling. Our prevascularization strategy may be useful to design large three-dimensional engineered tissues.

A three-dimensional in vitro model of angiogenesis in the airway mucosa.

Bronchial asthma is an inflammatory disease characterized by chronic intermittent bronchoconstriction. A key feature of the disease is structural changes in the airway wall (airway remodeling) consistent with tissue growth and chronic wound healing including angiogenesis. The epithelium directs mesenchymal processes during both embryogenesis and wound healing, and thus we hypothesized that the bronchial epithelium plays a critical role in directing angiogenesis. To study angiogenesis in the airways, we have developed a three-dimensional (3-D) in vitro model of the airway mucosa that consists of normal differentiated human bronchial epithelial cells (NHBE), normal human lung fibroblasts (NHLF), and human umbilical vein endothelial cells (HUVEC). The HUVEC are coated on dextran beads and suspended in a fibrin gel approximately 2mm beneath a confluent monolayer of NHLF which are just beneath the confluent monolayer of differentiated NHBE. In the presence of fibroblasts, visible capillaries reaching lengths of up to 1mm sprout from the HUVEC-coated beads. Over 11 days in culture, the bronchial epithelium produces transforming growth factor-beta2 (TGFbeta2, 60pg/ml), significantly increases vascular endothelial growth factor (VEGF) more than 6-fold to a concentration of 1.85ng/ml, but does not significantly impact total network formation. Exogenous TGFbeta2 stimulates VEGF production in a dose-dependent fashion (0-400pg/ml) through a MAPK-dependent pathway, but also inhibits capillary network formation. We conclude that the bronchial epithelium produces biologically relevant concentrations of VEGF and TGFbeta2 in a 3-D model of the airway mucosa that may be useful in probing mechanisms of angiogenesis in asthma.

Diffusion limits of an in vitro thick prevascularized tissue.

Although tissue engineering promises to replace or restore lost function to nearly every tissue in the body, successful applications are currently limited to tissue less than 2 mm in thickness. in vivo capillary networks deliver oxygen and nutrients to thicker (> 2 mm) tissues, suggesting that introduction of a preformed in vitro vascular network may be a useful strategy for engineered tissues. This article describes a system for generating capillary-like networks within a thick fibrin matrix. Human umbilical vein endothelial cells, growing on the surface of microcarrier beads, were embedded in fibrin gels a known distance (Delta = 1.8-4.5 mm) from a monolayer of human dermal fibroblasts. The distance of the growth medium, which contained vascular endothelial growth factor and basic fibroblast growth factor, from the beads, C, was varied from 2.7 to 7.2 mm. Capillaries with visible lumens sprouted in 2-3 days, reaching lengths that exceeded 500 microm within 6-8 days. On day 7, capillary network formation was largely independent of C; however, a strong inverse correlation with Delta was observed, with the maximum network formation at Delta = 1.8 mm. Surprisingly, the thickness of the gel was not a limiting factor for oxygen diffusion as these tissue constructs retained a relatively high oxygen tension of > 125 mmHg. We conclude that diffusion of oxygen in vitro is not limiting, allowing the development of tissue constructs on the order of centimeters in thickness. In addition, diffusion of fibroblast-derived soluble mediators is necessary for stable capillary formation, but is significantly impeded relative to that of nutrients present in the medium.