Modulated release from implantable ocular silicone oil tamponade drug reservoirs

ABSTRACT Complicated cases of retinal detachment can be treated with silicone oil tamponades. There is the potential for silicone oil tamponades to have adjunctive drug releasing behaviour within the eye, however the lipophilic nature of silicone oil limits the number of drugs that are suitable, and drug release from the hydrophobic reservoir is uncontrolled. Here, a radiometric technique was developed to accurately measure drug solubility in silicone oil and measure release into culture media. All‐trans retinoic acid (atRA), a lipophilic drug known to act as an anti‐proliferative within the eye, was used throughout this work. Chain‐end modification of polydimethylsiloxane with atRA produced a polydimethylsiloxane retinoate (PDMS‐atRA), which was used as an additive to silicone oil to modify the solvent environment within the silicone oil and the distribution coefficient. Blends of PDMS‐atRA and silicone oil containing different concentrations of free atRA were produced. The presence of PDMS‐atRA in silicone oil had a positive effect on atRA solubility and the longevity of release in vitro. The drug release period was independent of atRA starting concentration and dependent on the PDMS‐atRA concentration in the blend. A clinically relevant release period of atRA over 7 weeks from a silicone oil blend with PDMS‐atRA was observed. © 2018 The Authors. Journal of Polymer Science Part A: Polymer Chemistry Published by Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018, 56, 938–946


Experimental Details Characterisation
Nuclear Magnetic Resonance (NMR) spectra were recorded using a Bruker DPX-400 spectrometer operating at 400 MHz for 1 H NMR and 100 MHz for 13 C NMR. UV-Vis spectra were collected using a Thermo Fisher NanoDrop 2000c spectrophotometer with a quartz cuvette. Data was analyzed using the NanoDrop2000 software. All radiation measurements were carried out using a liquid scintillation counter (Packard Tri-carb 3100TR; Isotech). Triple detection gel permeation chromatography (GPC) was performed to measure molecular weights and molecular weight distributions using a Malvern Viscotek instrument. The instrument was equipped with a GPCmax VE2001 autosampler, two Viscotek T6000 columns (and a guard column), a refractive index (RI) detector VE3580 and a 270 Dual Detector (light scattering and viscometer) with a mobile phase of tetrahydrofuran (THF) containing 2 v/v % of trimethylamine at 35 °C with a flow rate of 1 mL min -1 .
A Zeiss Axiovert 200 microscope system was used to collect cell images. Fourier Transform Infrared (FT-IR) spectroscopy was carried out using a Bruker Tensor 27 plate reading FTIR. For each sample, 20 scans in the region from 650 to 4000 cm −1 were accumulated with a resolution of 4 cm −1 .

UV-Visible spectroscopy
A protocol reported in the literature was used; 1 a saturated solution of atRA in SIO was prepared and stirred gently in the dark at ambient temperature. Samples were taken at different time-points over a 2 week period and filtered using a syringe pump (4 mL/h) through 0.45 µm PTFE filters. The drug was extracted from the SIO three times with acetone (1:2 v/v). The acetone layers were combined and the solvent was evaporated with the remaining solid dried at ambient temperature. The samples were solubilised in 2 mL of a mixture of DMSO/H 2 O (8/2) and subsequently analysed by UV-Vis spectroscopy. A standard curve previously determined for atRA (see Electronic Supporting Information; ESI; Figure S1) was used to determine the drug concentration.

Cytotoxicity assays of drug compounds and atRA-PDMS-atRA
ARPE-19 cells between passage 8 and 24 were cultured at 37 °C in a dark, humid 5 % CO 2 incubator in media containing 1 % Pen-Strep, 1 % Amphotericin B and supplemented with 10 % FCS. For these studies, cells were used between passages 22 and 25. Multiple assays were carried out to study the effects of different drug concentrations on cell viability, metabolic activity and cell morphology. 18,000 cells/well were seeded in a 48 well tissue culture plate and left for 1 or 7 days to adhere to the plate. The 7 day samples were fed once within the week by replacing 450 µL old medium with 500 µL fresh culture medium. After the predetermined time period, the media was aspirated from all wells and replaced with 0.6 mL fresh media containing polymer blends or controls. Controls included: media, SIO 1000 (0.2 mL) and a positive control (20 v/v % DMSO in media). Cells were incubated for 1 to 7 days before the following assays could be performed. Resazurin stock solution was prepared by dissolving resazurin sodium salt in PBS at 0.1 mg/mL, filtered and stored at 4 °C in the dark. Medium was removed and replaced with resazurin solutions (10 % v/v stock solution in medium). Plates were incubated in the dark at 37 °C for 4 hours. The resazurin solution was removed and put in black 96-well plastic plates; resorufin fluorescence was read using a Biotek FLx800 spectrofluorometer (λExcitation = 530 nm; λEmission = 590 nm). All values were normalized to negative control wells on each plate. Finally statistical analyses were performed. Immediately following removal of resazurin solution, cells were washed with PBS (500 µL) then fixed for 10 minutes in 10 % neutral buffered formalin (NBF; 10 % formalin, approximately 4 % formaldehyde). NBF was discarded and the cells washed again with PBS. A phalloidin solution was used to stain the F-actin of the cytoskeleton of the cell. A phalloidin solution was produced at 1 mg powder in 1.5 mL MeOH according to manufacturer's instruction. The solution was then diluted 1 in 100 (in fresh PBS) and 75 µL was placed in each well followed by 30 minutes of incubation at 4 °C. Phalloidin solution was removed and the cells washed with PBS. DAPI was used to stain the nuclei of the cell. A stock solution of DAPI was made at 1:1,000 with PBS, then further diluted to a working solution of 1:10 PBS, 75 µL was placed in each well and incubated for 10 minutes at 4 °C. Cells were washed with PBS and placed under 500 µL PBS. Cells were then imaged using fluorescence microscopy.

Figure S14
1 H NMR spectra (CDCl 3 , 400MHz) of PDMS-atRA before and after exposure to KOH in THF at 40 °C for 22 h, displaying no change of the ester peak (2' and 3'), while the integration of the PDMS main chain signal diminished by a factor of 3-4.

Figure S15
GPC RI chromatogram overlays of PDMS-atRA (black) before and after exposure to KOH (2 and 5 equivalents) and NaOH (2 and 5 equivalents) in THF at 40 °C for 22 h.