Therapeutic designed poly (lactic-co-glycolic acid) cylindrical oseltamivir phosphate-loaded implants impede tumor neovascularization, growth and metastasis in mouse model of human pancreatic carcinoma

Figures & data

Figure 1 Therapeutic design of PLGA-OP cylindrical implants.

Notes: (A) Fabrication of PLGA-OP; (a, b) dissolved suspension of PLGA in acetone containing Span 80 with OP or without as a blank was transferred using a 1 mL glass syringe from a height of 4 cm onto a smooth Teflon sheet to form a flat, circular disk, in a fume hood for 1 hour followed by refrigeration at 5°C overnight; (c) PLGA-OP disk was lifted from the teflon sheet using a razor blade, and (d) rolled onto a glycerol lubricated precision glide 18 gauge syringe tip to form a cylinder; (e) fabricated PLGA-OP cylinders were extracted from the syringe needle and stored at −80°C; (f) surgical implantation of blank PLGA and PLGA-OP at tumor site (image showing PLGA-OP near necropsied live tumor at end point of experiment. (B) Photograph of PLGA-empty and PLGA-OP cylinders. (C) SEM micrographs of PLGA-OP cylinder; (a) micrograph of a single layer cross-section with hollow continuous center throughout the entire PLGA-OP structure, (b) top surface, (c) magnified porous surface structure, (d) magnified surface structure, and (e) magnified internal structure of PLGA-OP showing crystals of OP.
Abbreviations: OP, oseltamivir phosphate; PLGA, poly (lactic-co-glycolic acid); SEM, scanning electron microscope.

Figure 2 (A) A thermogram showing the glass transition temperatures of (a) PLGA polymer (32.7°C), PLGA loaded with (b) 20 mg (32.8°C), (c), 10 mg (15.2°C), and (d) 0 mg OP (5.8°C). (B) Cumulative release kinetics for PLGA blank, 10 mg and 20 mg OP loaded PLGA. All PLGA-OP samples exhibited a biphasic release profile.

Abbreviations: OP, oseltamivir phosphate; PLGA, poly (lactic-co-glycolic acid).

Figure 3 (A) PLGA-OP treatment of RAGxCγ double mutant mice bearing heterotopic xenografts of pancreatic PANC1 tumors. Cells at 0.5×10⁶ in 0.2 mL were implanted subcutaneously in the right back flank of these mice. Twice a week following implantation of the PANC1 cancer cells, each mouse was monitored for tumor volume ((width squared/2) × length) at the site of implantation. Mice were surgically implanted with sterilized PLGA-empty (four mice) and PLGA-OP (four mice cylinders) containing 20 mg OP at day 35 post-implantation of cancer cells when the tumor volume reached approximately 50–120 mm³. (B) Graph of mean ± SEM of live necropsy tumor weight per mouse body weight. Statistical analysis using unpaired t-test was carried out using GraphPad Prism, and the results were compared with untreated control cohort.

Abbreviations: OP, oseltamivir phosphate; PLGA, poly (lactic-co-glycolic acid); SEM, standard error of the mean; RAG, recombinase activating gene.

short-legendFigure 4

Notes: (A) Untreated control (three mice), (B) PLGA-empty (four mice), and (C) PLGA-OP (four mice) 20 mg treated cohorts. Necropsy tumors, HE staining of tumors, and paraffin-embedded tumor sections (5 μm) on glass slides were processed for immunohistochemistry using primary DyLight 488 conjugated rat monoclonal anti-mouse CD31⁺ (PECAM-1) antibody, primary anti-E-cadherin, and N-cadherin antibodies followed with polyclonal goat anti-rabbit Alexa Fluor^® 488 secondary antibody and Entellan^® rapid mounting media. Background control (not shown) sections were prepared without the primary antibodies and relative staining density was 2–4×10⁵. The bar on the stained tissue sections represents 200 μm. Images are representative of at least five fields of view from two tumor sections. (D) Quantitative analysis was done by assessing the density of tumor staining corrected for background in each panel using Corel Photo Paint 8.0 software. Each symbol in the figure represents the mean ± SEM corrected density of tumor staining within the respective images. Statistical analysis using unpaired t-test was carried out using GraphPad Prism and results were compared with the untreated cohort.
Abbreviations: OP, oseltamivir phosphate; PLGA, poly (lactic-co-glycolic acid); SEM, standard error of the mean.

Figure 5 Blank PLGA cylinders in culture with PANC1 cells. Morphology of viable cells at 3, 6, 10, and 15 days. Viability of cells was counted using 0.4% trypan blue solution and hemacytometer. Cell count was expressed as a percentage of control.

Abbreviation: PLGA, poly (lactic-co-glycolic acid).

Figure 6 Necropsy liver (A) and lung (B). RAGxCγ double mutant mice were implanted with 0.5×10⁶ PANC1 pancreatic cancer cells subcutaneously on the rear flank and PLGA-empty and PLGA-OP 20 mg cylinders were surgically implanted at day 35 post-implantation when tumors reached 100–120 mm³. Paraffin-embedded tissue sections (5 μm) on glass slides were processed for HE staining for each mouse necropsied at end point of the study. Stained tissue sections were photographed using a Zeiss Imager M2 fluorescence microscope at 400× magnification. Images are representative of at least five fields of view from two tissue sections. Metastatic tissue clusters were microscopically counted per tissue sections (5 μm) and plotted in the graph.

Abbreviations: OP, oseltamivir phosphate; PLGA, poly (lactic-co-glycolic acid); RAG, recombinase activating gene.