Spheronisation – PVA suspension and heating

1. Spheronisation – PVA suspension and heating • PLGA (85:15) 100-200 micron • Stirred 0.5 g in 50ml 0.2 % PVA in PBS at 80⁰C for 4 h • Top • Untreated • Middle • PEG 400 added (final 28% v/v) after 4 h • Bottom • No PEG added

2. Spheronisation – Rheology of spheronised PLGA microparticles +/- PEG 400 • PLGA (85:15) 100-200 micron • Stirred 0.5 g in 50ml 0.2 % PVA in PBSat 80⁰C for 4 h • Addition of PEG400 after 4h to “coat” particles

3. Spheronisation – PVA suspension and heating100-200 µm 6.5% PEG-PLGA 70 ⁰C in 0.2 % PVA in ddH2O 70 ⁰C C in 0.2 % PVA /6.5% PEG400 in ddH2O PEG 400 • Addition of PEG 400 leads to • Less swelling • Better spheronisation • Less agglomeration

4. Scaffold strength and porosity100-200 µm 6.5% PEG-PLGA - untreated or Spheronised for 4 h in 0.2 % PVA in ddH2O at 70 ⁰C

5. Biocompatability testing of carrier formulations • Assay setup • Linearity of plate reader response • linear to ODs ~ 3.0 • Linearity of cell response • linear to OD 1.1 • This response was from 4 x 105 cells/well 48 h after seeding (assay length) • OD ≥ 0.2 required by ISO10993-5. • OD > 0.4 for 1 x 105 cells/well 48 h after seeding.

6. Biocompatability testing of carrier formulations • Cytotoxicity testing • Addition of carriers directly to monolayers • 15 min, 1 h, 4 h and 24 h exposure. • Carrier aspirated and replaced with complete media after 15 min, 1 h and 4 h. • MTT assay carried out after 24h after initial exposure to carrier

7. Biocompatability testing of carrier formulations • Cytotoxicity testing • 2 % Pluronics F127 now used to improve injectability • No increased cytotoxic effects