Mechanical Control of Cell Proliferation Increases Resistance to Chemotherapeutic Agents.

While many cellular mechanisms leading to chemotherapeutic resistance have been identified, there is an increasing realization that tumor-stroma interactions also play an important role. In particular, mechanical alterations are inherent to solid cancer progression and profoundly impact cell physiology. Here, we explore the influence of compressive stress on the efficacy of chemotherapeutics in pancreatic cancer spheroids. We find that increased compressive stress leads to decreased drug efficacy. Theoretical modeling and experiments suggest that mechanical stress decreases cell proliferation which in turn reduces the efficacy of chemotherapeutics that target proliferating cells. Our work highlights a mechanical form of drug resistance and suggests new strategies for therapy.

Optimizing the Pharmacological Properties of Discoidal Polymeric Nanoconstructs Against Triple-Negative Breast Cancer Cells.

Fine-tuning loading and release of therapeutic and imaging agents associated with polymeric matrices is a fundamental step in the preclinical development of novel nanomedicines. Here, 1,000 × 400 nm Discoidal Polymeric Nanoconstructs (DPNs) were realized via a top-down, template-based fabrication approach, mixing together poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol)-diacrylate (PEG-DA) chains in a single polymer paste. Two different loading strategies were tested, namely the "direct loading" and the "absorption loading." In the first case, the agent was directly mixed with the polymeric paste to realize DPNs whereas, in the second case, DPNs were first lyophilized and then rehydrated upon exposure to a concentrated aqueous solution of the agent. Under these two loading conditions, the encapsulation efficiencies and release profiles of different agents were systematically assessed. Specifically, six agents were realized by conjugating lipid chains (DSPE) or polymeric chains (PEG) to the near-infrared imaging molecule Cy5 (DSPE-Cy5 A and DSPE-Cy5 B); the chemotherapeutic molecules methotrexate (DSPE-MTX and PEG-MTX) and doxorubicin (LA-DOX and DSPE-DOX). Moderately hydrophobic compounds with low molecular weights (MW) returned encapsulation efficiencies as high as 80% for the absorption loading. In general, direct loading was associated with encapsulation efficiencies lower than 1%. The agent hydrophobicity and MW were shown to be critical also in tailoring the release profiles from DPNs. On triple-negative breast cancer cells (MDA-MB-231), absorption loaded DOX-DPNs showed cytotoxic activities comparable to free DOX but slightly delayed in time. Preliminary in vivo studies demonstrated the high stability of Cy5-DPNs. Collectively, these results demonstrate that the pharmacological properties of DPNs can be finely optimized by changing the loading strategies (direct vs. absorption) and compound attributes (hydrophobicity and molecular weight).

A tissue chamber chip for assessing nanoparticle mobility in the extravascular space.

Although a plethora of nanoparticle configurations have been proposed over the past 10 years, the uniform and deep penetration of systemically injected nanomedicines into the diseased tissue stays as a major biological barrier. Here, a 'Tissue Chamber' chip is designed and fabricated to study the extravascular transport of small molecules and nanoparticles. The chamber comprises a collagen slab, deposited within a PDMS mold, and an 800 µm channel for the injection of the working solution. Through fluorescent microscopy, the dynamics of molecules and nanoparticles was estimated within the gel, under different operating conditions. Diffusion coefficients were derived from the analysis of the particle mean square displacements (MSD). For validating the experimental apparatus and the protocol for data analysis, the diffusion D of FITC-Dextran molecules of 4, 40 and 250 kDa was first quantified. As expected, D reduces with the molecular weight of the dextran molecules. The MSD-derived diffusion coefficients were in good agreement with values derived via fluorescence recovery after photobleaching (FRAP), an alternative technique that solely applies to small molecules. Then, the transport of six nanoparticles with similar hydrodynamic diameters (~ 200 nm) and different surface chemistries was quantified. Surface PEGylation was confirmed to favor the diffusion of nanoparticles within the collagen slab, whereas the surface decoration with hyaluronic acid (HA) chains reduced nanoparticle mobility in a way proportional to the HA molecular weight. To assess further the generality of the proposed approach, the diffusion of the six nanoparticles was also tested in freshly excised brain tissue slices. In these ex vivo experiments, the diffusion coefficients were 5-orders of magnitude smaller than for the Tissue Chamber chip. This was mostly ascribed to the lack of a cellular component in the chip. However, the trends documented for PEGylated and HA-coated nanoparticles in vitro were also confirmed ex vivo. This work demonstrates that the Tissue Chamber chip can be employed to effectively and efficiently test the extravascular transport of nanomedicines while minimizing the use of animals.

Modulating Phagocytic Cell Sequestration by Tailoring Nanoconstruct Softness.

The effect of nanoparticle size, shape, and surface properties on cellular uptake has been extensively investigated for its basic science and translational implications. Recently, softness is emerging as a design parameter for modulating the interaction of nanoparticles with cells and the biological microenvironment. Here, circular, quadrangular, and elliptical polymeric nanoconstructs of different sizes are realized with a Young's modulus ranging from ∼100 kPa (soft) to 10 MPa (rigid). The interaction of these nanoconstructs with professional phagocytic cells is assessed via confocal microscopy and flow cytometry analyses. Regardless of the size and shape, softer nanoconstructs evade cellular uptake up to 5 times more efficiently, by bone-marrow-derived monocytes, as compared to rigid nanoconstructs. Soft circular and quadrangular nanoconstructs are equally uptaken by professional phagocytic cells (<15%); soft elliptical particles are more avidly internalized (<60%) possibly because of the larger size and elongated shape, whereas over 70% of rigid nanoconstructs of any shape and size are uptaken. Inhibition of actin polymerization via cytochalasin D reduces the internalization propensity for all nanoconstruct types. High-resolution live cell microscopy documents that soft nanoconstructs mostly establish short-lived (<30 s) interactions with macrophages, thus diminishing the likelihood of recognition and internalization. The bending stiffness is identified as a discriminating factor for internalization, whereby particles with a bending stiffness slightly higher than cells would more efficiently oppose internalization as compared to stiffer or softer particles. These results confirm that softness is a key parameter in modulating the behavior of nanoparticles and are expected to inspire the design of more efficient nanoconstructs for drug delivery, biomedical imaging, and immunomodulatory therapies.

Deformable Discoidal Polymeric Nanoconstructs for the Precise Delivery of Therapeutic and Imaging Agents.

Over the last 15 years, a plethora of materials and different formulations have been proposed for the realization of nanomedicines. Yet drug-loading efficiency, sequestration by phagocytic cells, and tumor accumulation are sub-optimal. This would imply that radically new design approaches are needed to propel the clinical integration of nanomedicines, overcoming well-accepted clichés. This work briefly reviews the use of deformable discoidal nanoconstructs as a novel delivery strategy for therapeutic and imaging agents. Inspired by blood cell behavior, these nanoconstructs are designed to efficiently navigate the circulatory system, minimize sequestration by phagocytic cells, and recognize the tortuous angiogenic microvasculature of neoplastic masses. This article discusses the notion of nanoparticle margination and vascular adhesion, as well as advantages associated with deformable particles. Finally, details on the synthesis, physico-chemical properties, and in vivo characterization of discoidal polymeric nanoconstructs are provided, with particular emphasis on their ability to independently control size, shape, surface properties, and mechanical stiffness. These nanoconstructs could help in gaining a deeper understanding of the mechanisms regulating the behavior of nanomedicines and identifying optimal delivery strategies for patient-specific therapeutic interventions.