One of the major challenges in cancer therapy is ensuring delivery of chemotherapeutic agents to all cancerous cells. Suboptimal tissue penetration has been shown to be a factor limiting the effectiveness of several chemotherapeutic drugs including the anthracycline antibiotic doxorubicin. In this work, micellar formulations of doxorubicin were prepared, characterized and evaluated in 3-D, multicellular spheroid cultures. The multicellular spheroid cultures serve as an in vitro mimic of solid tumors. Poly(ethylene oxide) – poly(β-hydroxyalkanoic acid) – poly(ethylene oxide) triblock copolymers were synthesized as previously described and used to form micelles encapsulating doxorubicin. Doxorubicin-loaded micelles have also been shown to have a narrow size distribution with diameters of approximately 30-40 nm, and encapsulation of doxorubicin results in controlled drug release. We have demonstrated that unloaded polymeric micelles exhibit minimal cytotoxicity and that the cell-killing ability of doxorubicin is retained in micellar formulations. Furthermore, micelle-encapsulated doxorubicin has been found to inhibit the growth of multicellular spheroids and penetrate to the core of the spheroids more efficiently than free doxorubicin.

Mammalian cell culture systems are widely used in vitro models for the evaluation of drugs and drug delivery vehicles such as nanoparticles, mostly because they can help make subsequent animal tests more predictable and less costly. By and large, however, these systems have been formulated as 2D monolayers of cells which fail to take into account both extracellular delivery barriers and phenotypic differences of cells in vivo.  While nanoparticles delivered in a bulk solution to a 2D monolayer tend to reach and bind to cells with little interference, the same is not true for nanoparticles delivered in vivo to a 3D environment characterized by smaller pore sizes of the extracellular matrix (ECM).  Furthermore, cells in 2D monolayer cultures are exposed to a relatively uniform environment in culture medium, whereas cells grown in 3D tissue mimicking ECM gels are subject to gradients of pH, nutrients, and waste products that exert both stimulatory and inhibitory influences on cell phenotype and proliferation, much like cells in actual solid tumors. These differences between 2D and 3D cell culture systems highlight the need for 3D models that can more accurately capture in vivo conditions. The Pun lab has developed a 3D cell culture perfusion chamber that can be used to evaluate the penetration of fluorescently labeled nanoparticles into a cancer cell-hydrogel tumor mimic. Improvements have been made to the chamber system in regards to recovering live cells for post-experimental flow cytometry, a cell counting process to determine the percentage of cells associated with fluorescent nanoparticles and therefore the percent penetration into tissue. Recently, the system has been used to compare the penetration of polystyrene beads with that of pluronic micelles, the latter being a promising new vehicle for delivering anti-cancer agents to tumor tissue.