Tunable Collagen Microfluidic Platform to Study Nanoparticle Transport in the Tumor Microenvironment.

This chapter describes the motivation and protocol for creating a perfused 3D microfluidic in vitro platform representative of the tumor microenvironment to study nanoparticle transport. The cylindrical vascularized tumor platform described consists of a central endothelialized microchannel surrounded by a collagen hydrogel matrix containing cancer cells. This system can be employed to investigate key nanoparticle transport events in the tumor such as extravasation, diffusion within the extracellular matrix, and nanoparticle uptake. This easily manufactured tumor platform can be used for novel nanoparticle refinement focused on optimizing nanoparticle features such as size, shape, and functionalization method. This can yield ideal nanoparticles with properties that facilitate increased transport within the tumor microenvironment, leading to more effective nanoparticle-based treatments for cancer including nanoparticle-based drug delivery systems.

Single-walled carbon nanohorns decorated with semiconductor quantum dots to evaluate intracellular transport.

Single-walled carbon nanohorns (SWNHs) have great potential to enhance thermal and chemotherapeutic drug efficiencies for cancer therapies. Despite their diverse capabilities, minimal research has been conducted so far to study nanoparticle intracellular transport, which is an important step in designing efficient therapies. SWNHs, like many other carbon nanomaterials, do not have inherent fluorescence properties making intracellular transport information difficult to obtain. The goals of this project were to (1) develop a simple reaction scheme to decorate the exohedral surface of SWNHs with fluorescent quantum dots (QDs) and improve conjugate stability, and (2) evaluate SWNH-QD conjugate cellular uptake kinetics and localization in various cancer cell lines of differing origins and morphologies. In this study, SWNHs were conjugated to CdSe/ZnS core/shell QDs using a unique approach to carbodiimide chemistry. Transmission electron microscopy and electron dispersive spectroscopy verified the conjugation of SWNHs and QDs. Cellular uptake kinetics and efficiency were characterized in three malignant cell lines: U-87 MG (glioblastoma), MDA-MB-231 (breast cancer), and AY-27 (bladder transitional cell carcinoma) using flow cytometry. Cellular distribution was verified by confocal microscopy, and cytotoxicity was also evaluated using an alamarBlue assay. Results indicate that cellular uptake kinetics and efficiency are highly dependent on cell type, highlighting the significance of studying nanoparticle transport at the cellular level. Nanoparticle intracellular transport investigations may provide information to optimize treatment parameters (e.g., SWNH concentration, treatment time, etc.) depending on tumor etiology.

Flow measurements in a blood-perfused collagen vessel using x-ray micro-particle image velocimetry.

Blood-perfused tissue models are joining the emerging field of tumor engineering because they provide new avenues for modulation of the tumor microenvironment and preclinical evaluation of the therapeutic potential of new treatments. The characterization of fluid flow parameters in such in-vitro perfused tissue models is a critical step towards better understanding and manipulating the tumor microenvironment. However, traditional optical flow measurement methods are inapplicable because of the opacity of blood and the thickness of the tissue sample. In order to overcome the limitations of optical method we demonstrate the feasibility of using phase-contrast x-ray imaging to perform microscale particle image velocimetry (PIV) measurements of flow in blood perfused hydrated tissue-representative microvessels. However, phase contrast x-ray images significantly depart from the traditional PIV image paradigm, as they have high intensity background, very low signal-to-noise ratio, and volume integration effects. Hence, in order to achieve accurate measurements special attention must be paid to the image processing and PIV cross-correlation methodologies. Therefore we develop and demonstrate a methodology that incorporates image preprocessing as well as advanced PIV cross-correlation methods to result in measured velocities within experimental uncertainty.

Epigallocatechin-3-gallate (EGCG) attenuates inflammation in MRL/lpr mouse mesangial cells.

Epigallocatechin-3-gallate (EGCG), a bioactive component of green tea, has been reported to exert anti-inflammatory effects on immune cells. EGCG is also shown to activate the metabolic regulator, adenosine 5'-monophosphate-activated protein kinase (AMPK). Reports have also indicated that EGCG inhibits the immune-stimulated phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. The PI3K/Akt/mTOR pathway has been implicated in mesangial cell activation in lupus. Mesangial cells from MRL/lpr lupus-like mice are hyper-responsive to immune stimulation and overproduce nitric oxide (NO) and other inflammatory mediators when stimulated. In our current studies, we sought to determine the mechanism by which EGCG attenuates immune-induced expression of pro-inflammatory mediators. Cultured mesangial cells from MRL/lpr mice were pre-treated with various concentrations of EGCG and stimulated with lipopolysaccharide (LPS)/interferon (IFN)-gamma. EGCG activated AMPK and blocked LPS/IFN-gamma-induced inflammatory mediator production (iNOS expression, supernatant NO and interleukin-6). Interestingly, EGCG attenuated inflammation during AMPK inhibition indicating that the anti-inflammatory effect of EGCG may be partially independent of AMPK activation. Furthermore, we found that EGCG effectively inhibited the immune-stimulated PI3K/Akt/mTOR pathway independently of AMPK, by decreasing phosphorylation of Akt, suggesting an alternate mechanism for EGCG-mediated anti-inflammatory action in mesangial cells. Taken together, these studies show that EGCG attenuated inflammation in MRL/lpr mouse mesangial cells via the PI3K/Akt/mTOR pathway. Our findings suggest a potential therapeutic role for the use of EGCG to regulate inflammation and control autoimmune disease.

Deletion of PPAR-γ in immune cells enhances susceptibility to antiglomerular basement membrane disease.

Activation of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR-γ) has been shown to be immunoregulatory in autoimmune diseases by inhibiting production of a number of inflammatory mediators. We investigated whether PPAR-γ gene deletion in hematopoietic cells would alter disease pathogenesis in the antiglomerular basement membrane (anti-GBM) mouse model. PPAR-γ(+/+) and PPAR-γ(-/-) mice were immunized with rabbit antimouse GBM antibodies and lipopolysaccharide and evaluated for two weeks. Although both the PPAR-γ(+/+) and PPAR-γ(-/-) mice had IgG deposition in the glomerulus and showed proteinuria two weeks after injection, glomerular and tubulointerstitial disease in PPAR-γ(-/-) mice were significantly more severe compared with the PPAR-γ(+/+) animals. We observed that the PPAR-γ(-/-) mice had decreased CD4(+)CD25(+) regulatory T cells and an increased CD8(+):CD4(+) ratio as compared with the PPAR-γ(+/+) mice, suggesting that PPAR-γ has a role in the regulation of T cells. Furthermore, plasma interleukin-6 levels were significantly increased in the PPAR-γ(-/-) mice at two weeks as compared with the PPAR-γ(+/+) animals. Taken together, these studies show that the lack of PPAR-γ expression enhances inflammatory renal disease in the anti-GBM antibody-induced glomerulonephritis mouse model and suggests targeting PPAR-γ may have therapeutic efficacy.