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PEBCA NPs labeled with the lipophilic and fluorescent dye NR668 were used to study the biodistribution in MA98.12 bearing mice using an IVIS® Spectrum in vivo imaging system (Perkin Elmer). Mice were intravenously injected the same single dose PEBCA-CBZ or PEBCA without drug as in the efficacy study. The batch containing CBZ has somewhat larger particles than the batch not containing CBZ (Table S1). The excitation/emission wavelength pair of 535/640 nm was found to give the best signal-to-noise ratio and was thus used for imaging of the NPs. Whole body images were obtained 1, 4, 24 and 96 h after in-jection; the animals were then sacrificed by cervical dislocation and organs were harvested. The organs were imaged ex vivo with the IVIS scanner using the same settings as above. Relative signal intensity in the organs was calculated, using Living Image software (Perkin Elmer), as radiant efficiency (Emission light [photons/s/cm2/str]/ Excitation light [μW/cm2] × 10 ) per pixel of the region of interest, which was drawn around the respective organ. Fluorescent measurements of the PEBCA NPs used for in vivo imaging showed that the particles without CBZ had a fluorescent intensity of 1.17 times that of the PEBCA NPs containing CBZ and the data shown in Fig. 3C are corrected for this difference.
2.11. Biodistribution and pharmacokinetics in blood
Blood and tissue samples were obtained following a single i.v. in-jection of PEBCA-CBZ and non-encapsulated CBZ (15 mg/kg) into the tail vein of mice bearing the MAS98.12 tumor (n = 3). Empty particles (PEBCA) and saline were used as negative controls. The blood samples were taken either per tail vein puncture (approximately after 2 min and thereafter at 1, 4 and 24 h after injection) or by terminal cardiac puncture (24 and 96 h after injection) in Vacutainer tubes containing EDTA (BD Biosciences, San Jose, CA, USA) and kept on ice. The 0–24 h samples were collected consecutively from the same animals (n = 3) while samples after 96 h were obtained from separate mice (n = 3). The blood was centrifuged for 15 min at 4 °C and 3400 x g, and the super-natant plasma was collected and stored at −80 °C until LC-MS/MS analysis. The animals were sacrified after 24 or 96 h and tissue samples (tumors, livers, spleens, Tauroursodeoxycholate nodes and kidneys) were harvested. The organs were gently washed with saline and then snap frozen in liquid nitrogen and stored at −80 °C until further processing and LC-MS/MS analyses as described above. Statistical analyses were performed using the t-test.
Tumors from MAS98.12 bearing mice were collected 96 h after a single injection of the same substances as were injected in the MAS98.12 efficacy study. The tumors were preserved in 4% (v/v) for-malin and then paraffinized and sliced to prepare consecutive slides (3 μm thick). The deparaffinization agent Neo-clear and mounting agent Neo-mount were obtained from VWR (Radnor, PA, USA). Heat induced epitope retrieval was performed by placing deparaffinized slides with 10 mM sodium citrate buffer (pH 6.0) in water bath for 20 min at 100 °C. Endogenous peroxidase activity was blocked by incubating slides with 3% (v/v) hydrogen peroxide in Tris-buffered saline (TBS; 50 mM Tris-Cl, 150 mM NaCl, pH 7.6). Sections were washed and blocking of non-specific binding was performed with 3% (w/v) bovine serum albumin (Roche diagnostics GmbH, Mannheim, Germany) in TBS for 30 min. Sections were then incubated for 60 min with the primary antibodies. Three different antibodies, i.e. anti-CD68 (1 mg/ml; ab125212, Abcam, Cambridge, UK), anti-CD206 (0.1 μg/ml; ab64693, Abcam, Cambridge, UK), and anti-iNOS (0.5 μg/ml; ab15323, Abcam, Cambridge, UK) were used to detect different populations of Journal of Controlled Release 293 (2019) 183–192
macrophages. CD68 is a commonly used marker for the whole macro-phage population [20,21], iNOS (inducible nitric oxide synthase) is a marker for M1 macrophages (anti-tumorigenic and pro-inflammatory macrophages) , and CD206 is a marker for M2 macrophages (pro-tumorigenic and anti-inflammatory macrophages) . TBS was used for washing the slides between steps. Detection of primary antibodies was performed using MACH 3 rabbit HRP-polymer detection kit ac-cording to the manufacturer's protocol (Biocare Medical, Concord, CA, USA). Signals were developed by incubation with the Chromogen so-lutions provided with the Betazoid DAB Chromogen kit (Biocare Med-ical). For counter staining a haematoxylin and 37 mM ammonium hy-droxide containing solution (Sigma-Aldrich, St. Louis, MO, USA) were used.
Stained tissue sections were scanned (NanoZoomer HT, Hamamatsu Photonics, Hamamatsu, Japan) using a 40× objective. The extent of CD68, iNOS and CD206 was automatically scored using the ImmunoPath software (Room4 Ltd., Crowborough, UK) . The tumor areas were marked manually, excluding necrotic areas as well as blood vessels to avoid unspecific or false positive staining. The anno-tated areas were then broken down to smaller frames per image of in-terest for efficient processing. The annotated representative areas were analyzed further by computerized image analysis. The image analysis protocols were set up on randomly selected images from different tu-mors to educate the software to differentiate between haematoxylin stained blue negative staining and brown positive staining. The output of the analysis provides the number of the positive pixel fraction and the negative pixel fraction of total annotated area of the whole tumors.