Biodegradation of PLGA-MPs

Biodegradation of PLGA-MPs

Materials and reagents

1. SKH1-Elite mice (Charles River Laboratories Inc)
2. Poly (lactic-co-glycolic acid) (PLGA) microparticles
3. 18-gauge Monoject filter needle (Covidien, ordered through VWR, catalog #15141-198)
4. Methyl Cellulose, 4000 cP (Sigma Aldrich, product # M0512)
5. Scissors
6. Paraffin wax (Sigma Aldrich, product # 76228)
7. 70% ethanol solution
8. Glass jars
9. Tissue marking dye (Cancer Diagnostics Inc, product # 0726-2)
10. Pushpins
11. Glass slide (1 × 3 inch)
12. Hematoxylin and eosin

13. Formalin-free fixative (Fisherbrand™ Permaloc™ Formalin, #22-126-350)

14. Biopsy Cassettes  (Ariba,1212Y55)

Procedures 

1. Five empty PLGA microparticles were suspended in 50 uL of methyl cellulose (15 mg/ml; Sigma-Aldrich) solution.
2. Particles were aspirated into an 18-gauge Monoject filter needle (Covidien) and administered subcutaneously into the rear flank of SKH1-Elite mouse.
3. Mice were euthanized and their skins of the entire rear flank were harvested by scissors after 2, 8, and 30 days. The color of PLGA microparticles will turn from transparent to white over time after administration" to "  The color of PLGA microparticles turns from transparent to white over time after administration. 
4. The skin was then cut into ~1 cm×1 cm square containing PLGA microparticles, spread and pinned on a thin piece of wax (~1.5 cm×1.5 cm ×0.5 cm, length × width × thickness) using pushpins, fixed in formalin-free fixative (Sigma-Aldrich) for 24 hours, and transferred to 70% ethanol in a glass jar.
5. The nearest edge of the skin to PLGA microparticles was marked with tissue marking dye.
6. Skin samples were then embedded in paraffin. Subsequently, the embedded samples were sectioned using a microtome machine. Cross-sections measuring 5 μm in thickness were then cut from the side marked with tissue dye. The microtome was adjusted to 100 μm between each step. This process was repeated until reaching the depth necessary to visualize and analyze the PLGA microparticles.
7. The skin sections containing PLGA microparticles were then stained with hematoxylin and eosin stain and imaged using an Aperio AT2 Slide Scanner (Leica Biosystems).

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