The nanoparticles utilized in this experiment were dextran-coated, iron oxide (magnetite, Fe3
)-core BNF® nanoparticles with an average hydrodynamic diameter range of 100-130 nm (MicroMod GmBH, Rostock, Germany). The mean magnetite core diameter was approximately 45 nm. The nanoparticle iron concentration was 14.5 mg Fe/ml (33 mg nanoparticle/ml) in deionized water. The synthesis of these nanoparticles was described by Gruettner, et al [9
] and a complete description of their physical characteristics has been published [17
A murine breast adenocarcinoma cell line (MTG-B) [18
] was cultured in the Alpha modification of Eagle’s Minimal Essential Medium (MEM, HyClone Laboratories, Inc.) with 1% penicillin/streptomycin (Pen-Strep, HyClone Laboratories, Inc., Logan, UT, USA), 1% L-glutamine (Mediatech, Inc., Manassas, VA), and 10% fetal bovine serum (FBS, Hycolone Laboratories, Inc.) at 37 degrees Celsius in 5% CO2
atmosphere in an incubator (Queue Systems Inc., Parkersburg, VA, USA).
2.3 Animal Tumor Model
This experiment was approved by Dartmouth’s Institutional Animal Care and Use Committee and all animals were treated humanely, in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). To prepare cells for implantation in mice, the cells were exposed to trypsin (0.25% trypsin in EDTA, HyClone Laboratory, Inc.), stained with trypan blue (Hyclone Laboratories, Inc.), counted using a hemocytometer (Fisher Scientific Inc. Pittsburg, PA, USA), and then re-suspended in (serum-free, L-glutamine-free, Pen-Strep-free) Alpha MEM media at a concentration of 107 cells/mL. For each tumor, 100 μL of this solution was injected intradermally into the shaved flanks (one or two tumors per mouse) of female C3H/HE mice (The Jackson Laboratory, Bar Harbor, ME, USA), as described in . Three orthogonal tumor measurements were taken with a digital caliper every two days and the tumor volume was determined using the equation for the volume of an ellipsoid. When tumors reached volumes of greater than 50 mm3 they were considered of appropriate size for analysis.
This table shows the characteristics of each tumor used in the study.
Nanoparticles (5 mg Fe/cm3 tumor) were injected using a 30-gauge needle (0.34 μL of nanoparticle solution per mm3 of tumor). The tip of the needle was advanced into the center of the tumor and the nanoparticle suspension injected over the course of 30 seconds. The tip of the needle remained in place for five minutes post-injection to optimize nanoparticle distribution. Animals were euthanized and the tumors were excised, sectioned, fixed overnight, and processed for TEM at the pre-determined post-inject time endpoint ().
The number of tumors examined at each nanoparticle incubation time point.
2.4 Transmission Electron Microscopy
Tumor tissue samples were fixed in 200 μL of 4% glutaraldehyde (Ted Pella, Inc., Redding, CA, USA) solution overnight and transferred to 200 μL of 0.1 M sodium cacodylate buffer solution (pH 7.4, Ted Pella, Inc.) after three wash cycles with buffer. The samples were prepared for TEM (FEI Company Tecnai F20 FEG TEM operating at 100 kV) at the Dartmouth College Electron Microscopy Facility. Either L.R. White (Polysciences, Inc., Warrington, PA, USA) or Poly/Bed-812 (Polysciences, Inc.) was used as an embedding resin. Samples were stained with 4% osmium tetroxide (Ted Pella, Inc.) and en-bloc stained with 2% uranyl acetate (Ted Pella, Inc.) for one hour, each. Thin sections of 100-110 nm from each tumor sample were cut using a Leica Ultra-Cut Microtome (Leica Microsystems GmbH, Wetzlar, Germany).
2.5 Image Analysis to Quantify Uptake
To quantify nanoparticle uptake, computer code was written using Matlab 18.104.22.1684 (The Mathworks, Inc., Natick, MA, USA) Image Processing Toolbox. Ten low-magnification (5000x), randomly-chosen TEM fields from each tumor sample were digitally photographed. This magnification allowed for the assessment of multiple cells and associated extracellular spaces within one field of view. Due to electron density similarities—but morphologic differences— of nuclear chromatin and IONP, nuclei were manually excluded from images using a digital computer input tablet (Wacom Technology Corporation, Vancouver, WA, USA). Nanoparticles were never present within nuclei, so this exclusion did not affect quantification accuracy. Artifacts from sample preparation were also excluded. The images of the cells were then manually segmented along their plasma membranes in order to separately quantify internal and external IONP.
Due to the extreme election density of the IONP, we were able to segment each image to identify only IONP in a binary map of the intracellular region of interest (ROI). In the binary ROI, the nanoparticles were given a value of 0 and all other material a value of 1. This ROI map was inverted and summed to count the number of pixels corresponding to intracellular IONP. In a similar fashion, the extracellular space was analyzed using the same grayscale threshold value as the intracellular IONP. Once the pixels corresponding to nanoparticles were determined, an overlay was created to highlight the pixels determined to be nanoparticles and manual confirmation was completed. Using this analysis technique, the ratio of internal vs. external nanoparticles was quantified.
For each tumor sample (three tumors per time point) at times one, two, three, four, five and six hours, the total number of pixels corresponding to intracellular and extracellular nanoparticles was independently quantified. The percentage of intracellular vs. extracelluar IONP was then determined for each sample.