Preparation of dexamethasone-eluting nanoparticles
Initial attempts to replicate the method described by Agrahari et al . (Agrahari, 2017) to synthesise dexamethasone loaded nanoparticles using the penta-block co-polymer PGA-PCL-PEG-PCL-PGA (poly(glycolic acid)-poly (caprolactone)-poly (ethylene glycol)-poly (caprolactone)-poly (glycolic acid) were unsuccessful. In our hands we could not dissolve the co-polymer in dichloromethane, perhaps due to the crystallinity of high molecular weight poly-glycolic acid (Hacker and Mikos, 2011). Therefore, we chose to use a variant of the penta-block co-polymer described by Tamboli et al . (Tamboli, Mishra and Mitra, 2013), which was (poly(d,l )lactide-poly (caprolactone)-poly(ethylene-glycol)-poly (caprolactone)-poly(d,l )lactide (PDLLA-PCL-PEG-PCL-PDLLA).
Nanoparticles were synthesised from the PDLLA-PCL-PEG-PCL-PDLLA co-polymer using the oil-in-water solvent evaporation technique (McCall and Sirianni, 2013). 75 mg of penta-block co-polymer (AK099, PolySciTech, Akina Inc. West Lafayette, IN, USA) and 5 mg of dexamethasone (D4209, Sigma-Aldrich St. Louis, MO, USA) were added to 1.2 ml of ethyl acetate and vortexed into solution. The solution was added dropwise to 2 ml of vortexing 0.1% aqueous D-alpha-tocopherol polyethylene glycol 1000 succinate (E-TPGS, 57668, Sigma-Aldrich St. Louis, MO, USA). Immediately after mixing, the pre-emulsion was probe ultrasonicated (Branson 450 digital Sonifier, Branson Ultrasonic Corporation, CT, USA) in 10 x 30 second bursts at 20% power on ice. Between bursts, the emulsion was allowed to cool for 10 seconds. The emulsion was immediately added dropwise to 45 ml of 0.3% aqueous E-TPGS on a magnetic stirrer (300-400 rpm) for overnight solvent evaporation at room temperature. Nanoparticles were recovered by ultracentrifugation at 20,000 RPM for 1 hour at 4°C and washed 3 times with distilled water to remove E-TPGS and un-entrapped dexamethasone. Nanoparticles were lyophilized for 72 hours with trehalose, a lyoprotecterant (Zhouet al. , 2013), in a weight ratio of 0.5:1, trehalose:polymer (A19434.06, Alfa Aesar Haverhill, MA, USA), and stored at 4°C for further studies. Synthesis of unloaded nanoparticles was identical with the omission of dexamethasone. Average particle size was 167 nm [145, 189] (mean, [95% CI]) based on 150 measurements of individual particles in six scanning electron microscope images (JSM-5610, JEOL Ltd. Akishima, Tokyo, Japan) using the ‘measure’ function in ImageJ (Schneider, Rasband and Eliceiri, 2012).
We used mass spectrometry to measure the mass of dexamethasone contained within 5 mg of loaded nanoparticles. Nanoparticles were first dissolved in DMSO. An Agilent 6130 Quadrupole LC-MS coupled to an Agilent 1260 Infinity LC using a 150 X 4.6 mm Phenomenex Gemini NX-C18 column with a 110 Å pore size and 5 µm particle size was used to quantify dexamethasone concentration. Ultrapure water and acetonitrile, each containing 0.1% (v/v) formic acid (VWR) by volume, were used for the mobile phase at a flow rate of 1 ml/min. Samples were eluted with a gradient of 95% (v/v) water to 95% (v/v) acetonitrile over 10 minutes. The electrospray source was operated with a capillary voltage of 3.2 kV and a cone voltage of 25 V with nitrogen used as the nebulizer and desolvation gas at a total flow of 600 l/h. Detection was between 100-1000 Da in both positive and negative ionisation mode.
On the day of use, lyophilised nanoparticles were re-suspended in sterile PBS by bath sonication to a concentration of 0.25 mg/ml dexamethasone. Injections were performed under general anaesthesia induced by 5 minutes exposure to 4% isoflurane and 1 l/min oxygen in an anaesthetic chamber. Following loss of consciousness, confirmed by toe pinch, mice were transferred to a Bain co-axial circuit fitted with a mouse nose-cone to maintain anaesthesia. Mice received topical anaesthesia (Lidocaine hydrochloride 4%, Chauvin Pharma, Aubenas, France) immediately prior to injection of 20 µl nanoparticle suspension into the periocular tissue of each eye using a Hamilton syringe and a 30 gauge needle. Mice recovered in a 36°C chamber and were housed in groups of 5 for the remainder of the study.