Pullulan Based Bioconjugates for Ocular Dexamethasone Delivery

Abstract

Posterior segment eye diseases are mostly related to retinal pathologies that require pharmacological treatments by invasive intravitreal injections. Reduction of frequent intravitreal administrations may be accomplished with delivery systems that provide sustained drug release. Pullulan-dexamethasone conjugates were developed to achieve prolonged intravitreal drug release. Accordingly, dexamethasone was conjugated to ~67 kDa pullulan through hydrazone bond, which was previously found to be slowly cleavable in the vitreous. Dynamic light scattering and transmission electron microscopy showed that the pullulan-dexamethasone containing 1:20 drug/glucose unit molar ratio (10% w/w dexamethasone) self-assembled into nanoparticles of 461 ± 30 nm and 402 ± 66 nm, respectively. The particles were fairly stable over 6 weeks in physiological buffer at 4, 25 and 37 °C, while in homogenized vitreous at 37 °C, the colloidal assemblies underwent size increase over time. The drug was released slowly in the vitreous and rapidly at pH 5.0 mimicking lysosomal conditions: 50% of the drug was released in about 2 weeks in the vitreous, and in 2 days at pH 5.0. In vitro studies with retinal pigment epithelial cell line (ARPE-19) showed no toxicity of the conjugates in the cells. Flow cytometry and confocal microscopy showed cellular association of the nanoparticles and intracellular endosomal localization. Overall, pullulan conjugates showed interesting features that may enable their successful use in intravitreal drug delivery.

Keywords: controlled release; dexamethasone; hydrazone; ocular drug delivery; pullulan.

Scheme 1

Schematic representation of pullulan-dexamethasone derivative synthesis. Reagents and conditions: (I) ethyl bromoacetate 2, sodium hydride (NaH), anhydrous DMSO, room temperature (rt), two days (2d); (II) hydrazine hydrate 4, anhydrous DMSO, room temperature (rt), two days (2d); (III) dexamethasone 6, anhydrous DMF:DMSO, trifluoroacetic acid TFA, 40 °C, 2 days (2d); (IV a) NHS activated cyanine3 8, anhydrous DMF:DMSO, TEA, room temperature (rt), two days (2d); (IV b) NHS activated cyanine3 8, anhydrous DMSO, TEA, room temperature (rt), two days (2d).

Scheme 1

Schematic representation of pullulan-dexamethasone derivative synthesis. Reagents and conditions: (I) ethyl bromoacetate 2, sodium hydride (NaH), anhydrous DMSO, room temperature (rt), two days (2d); (II) hydrazine hydrate 4, anhydrous DMSO, room temperature (rt), two days (2d); (III) dexamethasone 6, anhydrous DMF:DMSO, trifluoroacetic acid TFA, 40 °C, 2 days (2d); (IV a) NHS activated cyanine3 8, anhydrous DMF:DMSO, TEA, room temperature (rt), two days (2d); (IV b) NHS activated cyanine3 8, anhydrous DMSO, TEA, room temperature (rt), two days (2d).

Figure 1

Fourier Transform–Infrared Spectrum of (A) pullulan: 3410 (-OH), 2928 (C-H), 1648 (C-O-C) cm⁻¹; (B) carboxyethyl-pullulan: 3402 (-OH), 2930 (C-H), 1736 (O-C=O), 1638 (C-O-C) cm⁻¹; (C) carboxyhydrazide-pullulan: 3400 (-OH), 2929 (C-H), 1655 (N-C=O), 1638 (C-O-C) cm⁻¹; (D) pullulan-dexamethasone: 3408 (-OH), 2926 (C-H), 1654 (-C=O-NH-N=C), 1637 (C-O-C) cm⁻¹. %T = Transmittance in percentage.

Figure 2

¹H NMR (400 MHz) spectra of (A) pullulan, (B) carboxyethyl-pullulan, (C) carboxyhydrazide-pullulan, (D) pullulan-dexamethasone and (E) dexamethasone in D₂O.

Figure 3

(A) Size distribution profile by intensity of pullulan-dexamethasone (■) and pullulan-dexamethasone-cyanine3 (■). (B) TEM images of pullulan-dexamethasone (left) and pullulan-dexamethasone-cyanine3 (right). Bar size: 500 nm.

Figure 4

Size (A) and poly dispersity index (PDI) (B) of pullulan-dexamethasone particles after different exposure times in buffer solution at 4 °C (■), 25 °C (■), 37 °C (■) and in homogenized vitreous at 37 °C (■).

Figure 5

Release profile of dexamethasone from pullulan-dexamethasone under three different conditions: PBS pH 7.4 (●); 1:1 v/v homogenized vitreous: PBS, pH 7.4 (■); phosphate-citric buffer, pH 5.0 (▲). Free dexamethasone in PBS, pH 7.4 (○) and in 1:1 v/v homogenized vitreous:PBS, pH 7.4 (□).

Figure 6

ARPE-19 cell viability profiles by MTT assays (A) after 24 h incubation with samples, and (B) after 24 h incubation followed by removal of samples and 48 h cell proliferation. Control are untreated cells (negative control, no polymeric conjugate or drug added), DMSO in the medium (0.8% v/v, one black squared symbol, ■), dexamethasone (positive control, ■) and carboxyhydrazide-pullulan as drug-free polymer (▲), pullulan-dexamethasone (▼) and fluorescently labeled pullulan-dexamethasone-cyanine3 (◆). The concentration of free dexamethasone or equivalent conjugated dexamethasone in pullulan-dexamethasone is plotted. The concentration of the carboxyhydrazide-pullulan was equivalent to that of pullulan in the cell samples treated with pullulan-dexamethasone.

Figure 7

Flow cytometric profile of ARPE-19 cells incubated with pullulan-dexamethasone-cyanine3 at increasing concentration. Concentration is referred to conjugated dexamethasone. MFI: Mean Fluorescence Intensity.

Figure 8

Confocal microscopic images of ARPE-19 cells incubated with 0.2 mg/mL of pullulan-dexamethasone-cyanine3 (PDC) nanoparticles in red. (A) Endosomes were stained in green with rabbit anti-EEA1 and Alexa Fluor 488 labeled goat anti-rabbit IgG H&L secondary antibody. (B) Lysosomes were stained in green with rat anti-mouse anti-LAMP1 and Alexa Fluor 488 labeled goat anti-rat IgG H&L secondary antibody. Nuclei were stained with DAPI in blue. RGB: overlay of red, green and blue channels for organelles/nanoparticles co-localization. (C) Magnification of the cell in the white square from (A) endosomes/nanoparticles from overlay (RGB). (D) Magnification of the cell in the white square from (B) lysosomes/nanoparticles from overlay (RGB). Bar size: 20 μm. (Note: bright field; RG: overlay of red and green channels are in SI-8, Figure S8).