The visualisation of vitreous using surface modified poly(lactic-co-glycolic acid) microparticles

Abstract

AIMS To demonstrate the potential use of in vitro poly(lactic-co-glycolic acid) (PLGA) microparticles in comparison with triamcinolone suspension to aid visualisation of vitreous during anterior and posterior vitrectomy. METHODS PLGA microparticles (diameter 10-60 microm) were fabricated using single and/or double emulsion technique(s) and used untreated or following the surface adsorption of a protein (transglutaminase). Particle size, shape, morphology and surface topography were assessed using scanning electron microscopy (SEM) and compared with a standard triamcinolone suspension. The efficacy of these microparticles to enhance visualisation of vitreous against the triamcinolone suspension was assessed using an in vitro set-up exploiting porcine vitreous. RESULTS Unmodified PLGA microparticles failed to adequately adhere to porcine vitreous and were readily washed out by irrigation. In contrast, modified transglutaminase-coated PLGA microparticles demonstrated a significant improvement in adhesiveness and were comparable to a triamcinolone suspension in their ability to enhance the visualisation of vitreous. This adhesive behaviour also demonstrated selectivity by not binding to the corneal endothelium. CONCLUSION The use of transglutaminase-modified biodegradable PLGA microparticles represents a novel method of visualising vitreous and aiding vitrectomy. This method may provide a distinct alternative for the visualisation of vitreous whilst eliminating the pharmacological effects of triamcinolone acetonide suspension.

Figure 1

Scanning electron microscope (SEM) and brightfield microscopy images of triamcinolone acetonide suspension and poly(lactic-co-glycolic acid) (PLGA) microparticles. Lyophilised triamcinolone solution and 20–100 μm PLGA microparticles were mounted on aluminium SEM stubs, gold-coated under an argon atmosphere before being imaged using a JOEL 6060LV variable pressure SEM operating at an accelerating voltage of 10 kV at the corresponding magnification (A, B and C, D, respectively). Brightfield images of 1 mg/ml triamcinolone solution and PLGA microparticles, in phosphate buffered saline (PBS), were achieved using a Leica DM-IRB/E inverted microscope and captured using the in-built imaging software (C and F, respectively).

Figure 2

Size distribution of fabricated poly(lactic-co-glycolic acid) (PLGA) microparticles. Microparticles were dispersed in 4 ml of distilled water within the chamber of a Coulter LS230 particle size analyser under moderate stirring. Particle size distribution was then determined as a function of the particle diffraction using the Coulter software (version 2.11a) and plotted, as shown above, as a function of the percentage of distribution volume.

Figure 3

Visualisation of vitreous using triamcinolone solution. The comparative visualisation of (A) control vitreous and (B) triamcinolone acetonide-treated vitreous were visualised using a Leica MZ16F stereomicroscope immediately following the addition of phosphate buffered saline (PBS) or triamcinolone (left) and following consecutive washes with PBS solution (right). Images were captured using the in-built software and shown at 1.6× magnification.

Figure 4

Visualisation of vitreous using poly(lactic-co-glycolic acid) (PLGA) microparticles. The comparative visualisation of vitreous following incubation with (A) untreated Oil-Red-O incorporated PLGA microparticles, (B) Oil-Red-O incorporated microbial transglutaminase-treated PLGA microparticles, (C) microbial transglutaminase-treated PLGA microparticles (no Oil-Red-O). Microparticles used were in the 20–100 μm diameter range and samples visualised using a Leica MZ16F stereomicroscope immediately following the addition (left) and after two consecutive washes (middle and right) of the microparticles with phosphate buffered saline (PBS). Images were captured using the in-built software and shown at 1.6× magnification.

Figure 5

Visualisation of vitreous using modified poly(lactic-co-glycolic acid) (PLGA) microparticles. The comparative visualisation of vitreous following incubation with (A) Fast-Green-FCF incorporated bovine serum albumin (BSA)-treated PLGA microparticles and (B) Oil-Red-O incorporated microbial transglutaminase-treated PLGA microparticles. Transglutaminase cross-linking activity was inhibited with the active-site directed inhibitor, R281, prior to adsorption on the microparticle surface. (C) Oil-Red-O incorporated mammalian transglutaminase-treated PLGA microparticles. Microparticles used were in the 20–100-μm range and samples visualised using a Leica MZ16F stereomicroscope following the addition (left) and after consecutive washes (middle and right) of the microparticles with phosphate buffered saline (PBS). Images were captured using the in-built software and shown at 1.6× magnification.