Polyvinyl alcohol (pva)

The CMP slurries used in this study were composed of 0.3 wt% super-fine wet ceria abrasives, a pH titrant (NaOH), a dispersant (PVA), and DI water. F.c.c. crystalline Ce(OH)₄ was synthesized as an abrasive particle with a primary particle diameter of ~ 2 nm. Ammonium cerium (IV) nitrate ((NH₄)₂Ce(NO₃)₃, Junsei Chemical, Tokyo, Japan) was used as the precursor material for the synthesis of Ce(OH)₄ abrasive particles. Additionally, imidazole (C₃H₄N₂, Sigma Aldrich, St. Louis, Missouri, USA) was used as a catalyst to improve the solubility of (NH₄)₂Ce(NO₃)₃. Sodium hydroxide (NaOH, Junsei Chemical, Tokyo, Japan) was used to adjust the pH. PVA (Polysciences, Warrington, US) was used as a dispersant. The slurry was prepared with the following concentration: 0.3 wt% nano-scale Ce(OH)₄ abrasives and 0.3 wt% PVA dispersant. The pH of the slurry was titrated at 6.0 using NaOH.

We used the following polymers: poly(ethylene glycol) (Sigma-Aldrich; MW  = 20000 Da), Ficoll (Sigma-Aldrich; MW  = 70000 Da and 400000 Da), dextran (Spectrum Chemical; 500000 Da), and poly(vinyl alcohol) (PVA) (Polysciences; MW  = 3000 Da)—formed by hydrolyzing 75% of poly(vinyl acetate). Solutions of AMPSs contained the following chemicals: ethylenediaminetetraacetic acid disodium salt (EDTA) (Sigma-Aldrich), potassium phosphate monobasic (EMD), sodium phosphate dibasic (Mallinkrodt AR), sodium chloride (EMD), MgCl₂ (USB), and Nycodenz (Axis-Shield PoC).

HeLa cells were cultured at 37 °C and 5% CO₂ in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, HyClone) supplemented with 10% (v/v) FBS (HyClone). Two days before imaging, cultured cells were plated onto plasma-etched coverslips (Fisher Premium Cover Glass, no. 1.5) spun coat with a 1% (w/v) polyvinyl alcohol (PVA, Polysciences Inc.) layer containing red (lamin B1) (580/605 nm, F8810, Invitrogen) or far red (mitochondria) (625/645 nm, F8806, Invitrogen) fluorescent microspheres, cultured for 24 h in high-glucose DMEM supplemented with 10% FBS, and subsequently cultured for 24 h in high-glucose, phenol-red-free DMEM (HyClone) supplemented with 10% FBS. During this period, some of the microspheres were endocytosed.

All reagents used in this study including the PLA were of analytical grade. Silicon wafers (SiO₂ substrate; KST World, Fukui, Japan) cut to an appropriate size (typically 3 × 3 cm) were treated with piranha solution, followed by washing with distilled water. PVA (Mw: 22 kDa; Kanto Chemical, Tokyo, Japan) was dissolved in distilled water at a concentration of 10 mg/mL, and this solution was dropped onto the SiO₂ substrates and spin-coated at 4,000 rpm for 20 s (Spin Coater MS-A100; Mikasa, Tokyo, Japan), followed by drying at 50 °C for 2 min. A solution of PLA (Mw: 80–100 kDa; Polysciences, Warrington, PA, USA) with the appropriate concentration (ca. 10 mg/mL), adjusted to the targeted thickness (150 nm), was dropped onto the PVA-coated substrates and spin-coated at 4,000 rpm for 20 s, followed by drying at 50 °C for 2 min. The obtained substrates were immersed in distilled water to collect free-standing nanosheets. The nanosheets were scooped up with coverslips and fully dried in a desiccator overnight. PDL or PDL with VTN-N was coated onto the PLA nanosheets immediately prior to use for culture experiments.

Brimonidine tartrate (BT)-loaded poly(lactic-co-glycolic) acid microspheres were fabricated as described previously, using a double emulsion procedure^{21 (link)}. Briefly, 200 mg of PLGA (MW 24–38 kDa, viscosity 0.32–0.44 dl/g) was dissolved in 4 ml of dicholoromethane (DCM). To this solution, 250 μl of a 50 mg/ml aqueous BT solution was added (prepared from solid BT, Santa Cruz Biotechnologies, Santa Cruz, CA). This suspension was then sonicated for 10 s and homogenized for 1 min in 2% poly(vinyl alcohol) (Polysciences) at 7000 rpm (Silverson L4RT-A). The emulsion was then mixed with a 1% PVA solution for 3 h to allow residual DCM to evaporate. The microspheres were then washed via centrifugation with deionized (DI) water prior to lyophilization for 48 hours (Virtis Benchtop K Freeze Dryer, Gardiner, NY). Dry microspheres were stored at −20 °C until use.
Drug-loaded microspheres were characterized for average size and surface morphology using volume impedance measurements (Multisizer 3 Coulter Counter, Beckman Coulter, Indianapolis, IN) and scanning electron microscopy (SEM, JEOL 6335 F Field Emission SEM, Peabody, MA). The volume average microsphere diameter was determined for a minimum of 10,000 microspheres. SEM images were also obtained for gel samples containing the microparticles (combined via passive mixing, as described below).